Method of treating a metabolic disorder of the liver

ABSTRACT

A method of treating a metabolic disorder of the liver includes administering a therapeutic agent. The therapeutic agent includes an IL-22 polypeptide and an antigen-binding molecule. The IL-22 polypeptide is fused or otherwise conjugated, directly or indirectly, to the antigen-binding molecule, and the antigen-binding molecule is an antibody or antigen-binding fragment that includes heavy and light chain CDR sequences.

PRIORITY AND CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 15/312,663, filed Nov. 20, 2016, which is the U.S. National StageApplication under 35 U.S.C. § 371 of International Application No.PCT/AU2015/050283, filed May 27, 2015, which claims priority to U.S.Provisional Application No. 62/003,492 entitled “Modulation of CellularStress” filed May 27, 2014, the contents of which are incorporatedherein by reference in their entirety.

SEQUENCE LISTING IN ELECTRONIC FORMAT

The present application is being filed along with an Electronic SequenceListing as an ASCII text file via EFS-Web. The Electronic SequenceListing is provided as a file entitled DAVI199022D1SEQLIST.txt, createdand last saved on May 19, 2020, which is 790,391 bytes in size. Theinformation in the Electronic Sequence Listing is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to compositions and methods formodulating oxidative and/or endoplasmic reticulum (OER) stress. Moreparticularly, the present invention provides compositions and methodsfor treating diseases associated with oxidative and/or OER stress,including metabolic disorders such as diabetes.

BACKGROUND OF THE INVENTION

Diabetes has rapidly developed into a major global epidemic. In 2011 itwas announced that 336 million people worldwide suffer from type 2diabetes, and this number is expected to grow to around 552 million by2030. Currently, the disease is responsible for approximately 4.6million deaths each year (Diabetes Atlas published by the InternationalDiabetes Federation). Such a high number of instances is creating anunsustainable financial burden to healthcare systems, with treatmentcirculating over USD$465 billion dollars in 2011, which is a significant11% of total healthcare expenditure in adults.

Despite current therapies, around 40% of individuals with type 2diabetes develop a requirement for exogenous insulin administration overtime, suggesting that these insulin secretory abnormalities progressafter the onset of the disease. Accordingly, there is an obvious andclear need to develop more effective therapies for the treatment of type2 diabetes, that improve the quality and/or quantity of insulinproduction, to address this gap in the current treatments.

Pancreatic B-cell Insulin Secretion

Glucose-induced insulin secretion by pancreatic β-cells is generallyschematized by a ‘consensus model’ that involves the following sequenceof events: acceleration of glucose metabolism; closure of ATP-sensitivepotassium channels (KATP channels) in the plasma membrane;depolarization, influx of Ca²⁺ through voltage-dependent calciumchannels and a rise in cytosolic-free Ca²⁺ concentration that inducesexocytosis of insulin-containing granules (Henguin et al., 2009).

Insulin resistance describes the state where insulin produces aninadequate biological effect, causing decreased insulin-induced muscleand adipose glucose uptake and increased hepatic glucose production.Subjects suffering from type 2 diabetes have some depletion of β-cellsand exhibit increased β-cell apoptosis (Butler et al., 2002). However,the extent of β-cell depletion is controversial and trials of bariatricsurgery and intense caloric restriction demonstrate that recovery ofinsulin production is possible in type II diabetics.

Sulfonylurea drugs have been a popular strategy for the development ofnovel treatments for type 2 diabetes over the last 60 years. This classof drug works by binding directly to the KATP channel, and thus inducingthe secretion of insulin from the 13-cells.

Endoplasmic Reticulum (ER) Stress

The ER is a major protein-folding compartment in a eukaryotic cell andis second only to the cytosol, and is the site of synthesis for allsecretory and cell surface proteins. Protein folding in the ER is morecomplex than protein folding in the cytosol because proteins arepost-translationally modified. Folding in the ER must coupleprotein-synthesis pathways operating outside of the compartment withER-assisted folding (ERAF) pathways in the lumen. Expression of a mutantversion of a protein, or even some-wild-type proteins, viral infection,energy or nutrient depletion, extreme environmental conditions, orstimuli that elicit excessive calcium release from the ER lumencompromise protein-folding reactions in the ER, causing unfolded proteinto accumulate and initiate a cascade of signals that are transmitted tothe cytoplasm and nucleus. When the protein-folding demand on the ERexceeds the folding capacity of the ER a condition termed “ER stress”results (Malhotra et al., 2007). ER stress can also result from otherconditions, namely when a protein that is translated in the cytosol ismisfolded and induces the recruitment of the folding machinery, leadingto a deficit of folding assistance in the ER.

The overproduction of secretory proteins has been demonstrated togenerate ER stress and it is well known that pancreatic islet β-cellssustain a high rate of insulin production and secretion. ER stressoccurs when protein-misfolding increases beyond threshold levels,releasing a suite of signaling and transcriptional events knowncollectively as the unfolded protein response (UPR). The ultimate goalof the UPR is to restore ER protein biosynthesis, but paradoxically oneinitial step is a suppression of translation of the secretory proteins,such as insulin in the β-cell, to relieve ER load. Accordingly, thephysical response mechanism, whilst protecting the secretory cell, canhave adverse physiological effects. For example, in β-cells this wouldbe a reduction in insulin production and, hence, hyperglycemia. Bothβ-cell ER stress and insulin misfolding have been reported in both type2 diabetes and autoimmune type 1 diabetes.

β-cells are long-lived cells that require a highly developed ER in orderto produce extremely large amounts of insulin over a long period oftime. This insulin generation requires a complex series of molecularbiosynthetic events that are initiated in the ER. Pre-pro-insulin, theprecursor of mature insulin, is cotranslationally translocated in the ERlumen, where the signal sequence is cleaved and proinsulin is generated.Machinery enzymes in the ER catalyze the formation of three disulfidebonds, to help form its native shape, before being trafficked to theGolgi apparatus and secretory granules. Finally, in the downstream Golgiit is further processed by endoproteases and the C-peptide is removed,generating the final product, mature insulin.

Potential contributors to β-cell ER stress in type 2 diabetes includeincreased insulin biosynthesis, lipotoxicity due to saturated long chainfree fatty acids (FFA) such as palmitate (a side effect of obesity),increased reactive oxygen species (ROS) production in the ER due toincreased disulfide bond formation and by mitochondria due to highglucose, and progressive accumulation of amyloid deposits (Cnop et al.,2012, Cunha et al., 2008).

The precise molecular mechanisms governing how insulin resistanceinterrelates to β-cell dysfunction are currently not well understood,with many obese individuals being highly insulin resistant withoutdeveloping β-cell dysfunction and diabetes. However, insulin resistanceadds to the demand for increased insulin biosynthesis in β-cells.Furthermore, the inappropriate secretion of the proinsulin precursor byβ-cells, which increases as diabetes progresses, is thought tocontribute to increased insulin resistance. Loss of cyclicity of insulinrelease from the pancreas also occurs in diabetes and contributes toincreasing insulin resistance. Oxidative stress, ER stress, amyloiddeposition in the pancreas, as well as both lipotoxicity andglucotoxicity are believed to play a role in progressive β-celldysfunction, and are all caused by over-nutrition.

IL-22 Treatment

Huang et al. (U.S. 2010/0015086) have shown that injection ofrecombinant IL-22 (rIL-22) can reduce total serum glucose, triglycerideand insulin levels as well as body weight in obese mice. They have alsoshown that systemic administration of rIL-22 can improve glucosetolerance and insulin sensitivity in normal mice. Based on theseobservations, Huang et al. propose that rIL-22 would be useful fortreating metabolic disorders including obesity, diabetes,hyperlipidemia, hyperglycemia and hyperinsulinemia. However, Huang etal. do not disclose any mechanism of action to explain how rIL-22achieves these effects.

The present inventors observe, however, that no clinical trials havebeen published that support the use of systemic administration of rIL-22for treating metabolic disorders in humans since publication of Huang etal. and that this may be due to possible off-target effects of rIL-22.For example, chronic high concentrations of IL-22, and lowconcentrations of the counteracting IL-22 binding protein (Dumoutier etal., 2001), have been shown to contribute to hyperplasia and tumordevelopment in the intestine in animal models (Huber et al., 2012;Kirchberger et al., 2013; Yu et al., 2013; Jiang et al., 2013). IL-22has also been reported to induce hepatocyte proliferation andoverexpression of IL-22 in the liver has been shown to increase the rateof carcinogen-induced liver cancers (Park et al., 2011). It has alsobeen reported that IL-22 is associated with human hepatocarcinomas, andthat carcinogen-induced liver cancers are reduced in IL-22 knockout mice(Jiang et al., 2011). IL-22-producing CD8 T cells have been foundassociated with transplant-associated skin cancers and possiblycontribute to squamous cell carcinoma growth or development in thissetting (Zhang et al., 2013). As shown herein, the present inventorshave also discovered that systemic administration of rIL-22 has thepotential to exacerbate viral infections in humans.

SUMMARY OF THE INVENTION

The present invention arises, in part, from the unexpected discoverythat specific cytokines including IL-22 inhibit oxidative andendoplasmic reticulum (OER) stress in pancreatic β-cells, whist othersincluding IL-23 and IL-24 actively increase it. In particular, thepresent inventors found that blockade of IL-23 or IL-24 improvespancreatic ER function, insulin biosynthesis and glucose tolerance. Bycontrast, they found that IL-22 is a powerful endogenous suppressor ofβ-cell OER stress in response to cytokines, lipids and proteinmisfolding agents. Significantly, IL-22 administration to obese animalswas found to modulate oxidative stress regulatory genes, reduce ERstress and promote secretion of high quality efficacious insulin, whichrestores glucose homeostasis before subsequent restitution of peripheralinsulin sensitivity. Based on these findings and their observations thatrIL-22 may have significant side effects when administered systemically,the present inventors propose that targeting β-cell OER stressinhibitors such as IL-22 directly to pancreatic β-cells will be usefulfor treating diseases associated with aberrant β-cell OER stress,including diabetes and other metabolic disorders, with reducedoff-target effects, as described hereafter.

Accordingly, in one aspect, the present invention provides therapeuticagents, which are suitably useful for reducing OER stress in apancreatic β-cell (hereafter referred to as “β-cell”). These therapeuticagents generally comprise, consist, or consist essentially of a β-cellOER stress inhibitor and a targeting agent that targets the inhibitor toa β-cell. In specific embodiments, the β-cell OER stress inhibitor isselected from the group consisting of: an IL-22 polypeptide, an IL-10polypeptide, or an antagonist of IL-23, IL-24, IL-33, IL-1β, MIP-2a,IL-17A, IFN-γ or IFN-8. The β-cell OER stress inhibitor is preferably anIL-22 polypeptide. In other embodiments, the β-cell OER stress inhibitoris selected from antagonists of IL-23 or IL-24.

Suitably, the targeting agent binds a β-cell protein (e.g., a β-cellsurface protein). In some embodiments, the β-cell protein is a β-cellreceptor (e.g., sulfonylurea receptor 1 (SUR1), glucagon-like peptide 1receptor (GLP-1R) or a G-protein-coupled receptor (e.g., GPR40, GPR119).The targeting agent is suitably selected from proteinaceous molecules(e.g., peptides, polypeptides) and small molecules.

In some embodiments, the targeting agent is a SUR1 ligand. The SUR1ligand is suitably a SUR1 agonist. Representative SUR1 ligands includeα-endosulfine, an antigen-binding molecule that is immuno-interactivewith SUR1, and sulfonylurea compounds, illustrative examples of whichinclude carbutamide, acetohexamide, chloropropamide, tolbutamide,tolazamide, glipizide, gliclazide, glibenclamide, glibornuride,gliquidone, glisoxepide, glyclopyramide, and glimepiride.

In other embodiments, the targeting agent is a GLP-1R ligand. Suitably,the GLP-1R ligand is a GLP-1R agonist. Non-limiting examples of GLP-1Rligands include glucagon-like peptide 1 (GLP-1), exendin-4, gastricinhibitory polypeptide, glucagon, taspoglutide, liraglutide, anantigen-binding molecule that is immuno-interactive with GLP-1R, andsmall molecule ligands such as hydroxylflavonol compounds (e.g.,quercetin).

In still other embodiments, the targeting agent is a G-protein-coupledreceptor (e.g., GPR40 or GPR119) ligand. Illustrative examples of thistype include AMG-837, TAK-875, LY2881835, HD0471042 and HD0471953.

In still other embodiments, the targeting agent is an antigen-bindingmolecule that is immuno-interactive with a β-cell protein (e.g., aβ-cell surface protein), which may or may not be characterized.

The targeting agent is suitably attached to the β-cell OER stressinhibitor either directly or indirectly (e.g., via an intervening linkerto the N-terminus or the C-terminus of the β-cell OER stress inhibitor).Alternatively, the targeting agent and the β-cell OER stress inhibitorare attached to or otherwise comprised in a delivery vehicle (e.g., aparticle, a dendrimer or a cyclodextrin).

In another aspect, the present invention provides pharmaceuticalcompositions that generally comprise a therapeutic agent as broadlydescribed above and elsewhere herein, and a pharmaceutically acceptablecarrier, excipient or diluent.

Yet another aspect of the present invention provides methods forinhibiting or reducing OER stress in a β-cell. These methods generallycomprise, consist or consist essentially of contacting the β-cell with atherapeutic agent as broadly described above and elsewhere herein suchthat the targeting agent binds to the β-cell and the β-cell OER stressinhibitor inhibits or reduces OER stress in the β-cell.

In still another aspect, the present invention provides methods oftreating a metabolic disorder in a subject. These methods generallycomprise, consist or consist essentially of administering to the subjectan effective amount of a therapeutic agent as broadly described above orelsewhere herein. Representative metabolic disorders includepre-diabetes, diabetes (type I or type II), metabolic syndrome, obesity,non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis(NASH), diabetic dyslipidemia, hyperlipidemia, hypertension,hypertriglyceridemia, hyperfattyacidemia, and hyperinsulinemia.

In some embodiments, the methods further comprise administeringconcurrently to the subject at least one ancillary agent for treatingthe metabolic disorder. In specific embodiments, the at least oneancillary agent is selected from an antidiabetic agent (e.g., metformin,glyburide, glimepiride, glipyride, glipizide, chlorpropamide,gliclazide, acarbose, miglitol, pioglitazone, troglitazone,dapagliflozin, rosiglitazone, insulin, GI-262570, isaglitazone, JTT-501,NN-2344, L895645, YM-440, R-119702, A39677, repaglinide, nateglinide,KAD1129, APR-H039242, GW-409544, KRP297, AC2993, Exendin-4, LY307161,NN2211 or LY315902), an anti-obesity agent (e.g., Orlistat, ATL-962,A39677, L750355, CP331648, sibutramine, topiramate, axokine,dexamphetamine, phentermine, phenylpropanolamine, famoxin, or mazindol)or a lipid-modulating agent (e.g., pravastatin, lovastatin, simvastatin,atorvastatin, cerivastatin, fluvastatin, nisvastatin, ZD-4522,fenofibrate, gemfibrozil, clofibrate, implitapide, CP-529,414,avasimibe, TS-962, MD-700, or LY295427).

The therapeutic agents and methods of the present invention are usefulfor promoting insulin secretion from a β-cell, improving the quality ofinsulin produced from a β-cell and/or slowing or reducing β-celldegeneration, and/or for promoting β-cell regeneration, includingfollowing pancreatic islet transplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Cytokines induce ER stress in murine pancreatic β-cells. (A)ERAI-XBP1-reporter fluorescence in MIN6N8 murine insulinoma β-cellsafter 24 hour treatment with 16.6 mM glucose (Hi glucose), 10 μM H₂O₂,0.5 mM palmitic acid or 50 ng/mL cytokines, or 6 hour treatment with 10μg/mL tunicamycin (Tm) or 5 μM thapsigargin (Tg). Right panel showsdose-response curves for stressor cytokines from 2-64 ng/mL. (B) MIN6N8cells exposed to Tm, Hi glucose or cytokines as in (a) and qRT-PCR usedto assess splicing of the Xbp1 mRNA, transfected with ATF6-GFP andnuclear translocation of activated ATF6 quantified, or an ALPHASCREENassay used to assess eIF2a phosphorylation. (C) Peak production ofreactive oxygen species (ROS) and nitric oxide (NO) in MIN6N8 cellstreated as in (A) from a 30 min to 8 hour time course. (D) ER stressinducing cytokines progressively activate the unfolded protein response.MIN6N8 murine β-cells were transfected with the ERAI reporter andexposed to tunicamycin (Tm, 10 μg/mL), IL-23 or IL-24 (50 ng/mL) and GFPfluorescence assessed at regular intervals over 36 hours. MIN6N8 vehiclecontrol (Con)=DMSO. Box-plots show median/IQR/range; histograms showmean±SEM; ANOVA, Bonferroni post-hoc test; *P<0.05, **P<0.01, ***P<0.001(from post-hoc test); * vs untreated vehicle control.

FIG. 2: IL-22 suppresses β-cell ER stress. (A-D) ERAI-XBP1-reporterfluorescence in MIN6N8 cells. (A) Heat map of ERAI-reporter fluorescencein MIN6N8 cells exposed to pair-wise combinations of cytokines, or a 6hour treatment with 10 μg/mL tunicamycin (Tm). Inset shows IL-10 andIL-22 dose-response for suppression of IL-23-induced ER stress. (B,C)Cells were exposed to 0.1-0.5 mM palmitic acid (B) or 5 μM thapsigargin(Tg) (C)±IL-22 for 24 or 6 hours, respectively. (D, left panel) Cellswere treated with IL-22 or Tm±inhibitors for STAT1 (FLUDARABINE) and/orSTAT3 (VI S31-201) for 6 hours. (D, right panel) MIN6N8 cells weretreated with Tm±IL-22±anti-IL-22R1 antibody for 6 hours and Xbp1splicing assessed. (E) IL-22R1 expression determined byimmunofluorescence and confocal microscopy in pancreata from mice fednormal chow or a high fat diet for 22 weeks; scale bar=25 μm.

FIG. 3: IL-22 suppresses β-cell oxidative stress. (A) MIN6N8 cellsloaded with dihydroethidium (DHE) which fluoresces red and binds DNAafter oxidation were exposed to 50 ng/mL IL-23 for 30 min±50 ng/mL IL-22at the same time or 30 min prior to IL-23. DAPI staining (blue) used tovisualize nuclei. Confocal micrographs after 30 min exposure; scalebar=100 μm. (B,C) Concentration of ROS (B) and NO (C) in MIN6N8 cellstreated with 16.6 mM glucose (Hi glucose), 10 μM H₂O₂, 0.5 mM palmiticacid or 50 ng/mL cytokines±50 ng/mL IL-22 administered 30 min before orat the same time as the stressors. Data shown are at the peak time ofproduction in non-IL-22-treated cells (30 min, 2 hours or 8 hours).Color reproductions of FIG. 3 are available upon request.

FIG. 4: Cytokines induce reactive oxygen species (ROS) and nitric oxide(NO) production in β-cells. MIN6N8 murine β-cells were exposed tocytokines, H₂O₂ or palmitate. ROS (shown in grey circles) was assessedby DCFDA method and nitrite (NO, black circles) was measured by usingthe Griess assay. IL-22 pretreatment markedly reduced the production ofROS (grey triangles) and NO (black triangles). Note the y-axes havediffering scales for different stressors.

FIG. 5: Assessment of oxidative stress genes influenced by IL-22. (A)MIN6N8 cells were treated with DMSO (control) or IL-22 and analyzedusing Oxidative Stress RT2 Profiler PCR Array. Genes upregulated (abovethe 2-fold upregulated line) or downregulated (below the 2-folddownregulated line) are shown above. (B) Alteration in the expression ofoxidative stress genes was confirmed by qRT-PCR in MIN6N8 cells treatedwith DMSO (control) or IL-22. (C) List of genes in the array that wereand were not regulated by IL-22.

FIG. 6: Cytokine-regulated oxidative and ER stress affects islet insulinsecretion. sXbp1and Hspa5mRNA (A), insulin (B) and proinsulin (C)secretion from healthy islets cultured for 6 hours in tunicamycin (Tm)or thapsigargin (Tg)±IL-22 or ER stress-inducing cytokines±IL-22 andthen cultured for another 18 hours before assessing glucose-stimulatedinsulin secretion as follows: islets were cultured consecutively for 30min each with 2.8 mM glucose, 20 mM glucose, and 20 mM glucose+100 nMGLP-1 and total insulin (B) and proinsulin (C) secretion measured byELISA.

FIG. 7: Endogenous and exogenous IL-22 affects healthy islets and isletsfrom obese mice. (A) mRNA expression of oxidative and ER stress genes inhealthy islets cultured for 24 hours in control (IgG) or αIL-22R1antibodies, and in islets from mice on a high fat diet (HFD), afterculturing for 24 hours in control IgG or IL-22. (B) Total insulinsecretion in 30 min by healthy and HFD islets cultured±glucose (“Higlu”) following culture in glucose±IL-22, IgG or αIL-22R1.

FIG. 8: Inflammation and oxidative stress gene expression in murineobesity and diabetes. Cytokine and chemokine gene (A, C) and oxidativestress regulatory gene (B, D) mRNA expression in islets from mice fednormal chow (Con) or a high fat diet (HFD) (A, B), and leptin receptordeficient (db/db) vs heterozygous (dbh) littermates (C, D). (E) Cytokineand cytokine receptor genes relevant to oxidative and ER stress withinthe 100 most highly upregulated genes (ranking shown) in a study ofhuman type 2 diabetes islets versus healthy islets.

FIG. 9: Targeting cytokine-induced ER stress in HFDIO improves glucosetolerance and hyperinsulinemia. HFDIO Experiment 1: Mice were fed a highfat diet (HFD) or normal chow diet (NCD) for 16 weeks and treated forthe last 3 weeks with (A-C) IL-22, (D-F) anti-IL-24, or (G-I)anti-IL-23. NCD and HFD control mice received an irrelevant IgG controlantibody. Blood glucose (A, D, G) and serum total insulin (C, F, I)during fasted i.p. glucose tolerance tests on day 14 of treatment, andblood glucose during insulin tolerance tests on day 16 (B, E, H) areshown. Fasting serum proinsulin:insulin ratio (3) and total pancreaticsXbp1 mRNA (K) at the end of treatment. (L) Co-staining of total insulinand Grp78 analyzed by confocal-microscopy (upper panels) andimmunohistochemical staining of proinsulin (lower panel) in pancreatafrom control and IL-22-treated mice. Graphs show quantification ofGrp78, total insulin and proinsulin in the core β-cell rich area ofislets. Scale bars=50 μm. Color reproductions of FIG. 9 are availableupon request.

FIG. 10: Neutralizing ER stress inducing cytokines or treating withIL-22 reduces total pancreatic ER stress but does not effect insulingene expression in HFDIO. (A, B) Mice were fed a high fat diet or normalchow for 16 weeks and treated for the last 3 weeks with anti-IL-23,anti-IL-24, or IL-22 or an irrelevant control antibody (Con) (asdescribed for FIG. 9). Pancreatic ER stress assessed by measurement ofHspa5 (Grp78) (A) and insulin (Ins2) (B) gene expression assessed byqRT-PCR after 3 weeks treatment. (C-E) Mice were fed a HFD or NCD for 22weeks and treated for the last 30 days with IL-22 at a concentration ofeither 20 or 100 ng/g (as described in FIG. 5: HFDIO Experiment 2).Insulin (Ins2) (C) and MafA (D) mRNA expression assessed in islets fromcontrol and treated mice. (E) Similar to insulin mRNA levels, no changeswere observed in the prohormone convertase PC1(PC3) in the HFD micetreated with IL-22. (A-D) Normalization against the housekeeping geneGapdh, and expression as fold difference to the untreated normal chowcontrol mice.

FIG. 11: Assessment of ER stress in pancreatic α- and β-cells in micewith HFDIO with and without treatment with IL-22. Mice were fed a highfat diet for 16 weeks and treated for the last 3 weeks with anti-IL-23,anti-IL-24 or IL-22 (as per FIG. 9) legend. Paraffin sections ofpancreata were prepared and co-stained with antibodies against the ERstress marker Grp78, and the α-cell marker glucagon (A) or the β-cellmarker insulin (B). Individual single color low power images are shownat the left, and dual color low and high power images are shown at theright. Quantification of Grp78 and insulin in β-cells is shown in FIG.9. White arrows highlight the acinar cells surrounding the islets.

FIG. 12: Neutralizing ER stress inducing cytokines or treating withIL-22 reduces total pancreatic inflammation in HFDIO. In HFDIOExperiment 1 mice were fed a high fat diet for 16 weeks and treated forthe last 3 weeks with anti-IL-23 (A), anti-IL-24 (B), or IL-22 (C) or anirrelevant control antibody (as per FIG. 9). Pancreatic cytokine geneexpression was assessed qRT-PCR with normalization against thehousekeeping gene Gapdh, and expression as fold difference to theuntreated HFDIO mice.

FIG. 13: Altered food consumption does not explain loss of body weightin IL-22-treated HFDIO mice. HFDIO experiment 1 as described for FIG. 9.(A) Body weight changes in HFDIO experiment 1. (B) Results are presentedas food consumed per mouse per week. Food consumption in the last 2weeks of treatment during experiment 1. Mean±SEM.

FIG. 14: Neutralizing ER stress inducing cytokines or treating withIL-22 reduces hepatic upregulation of Grp78 in HFDIO. As described forHFDIO experiment 1 mice were fed a high fat diet or normal chow for 16weeks and treated for the last 3 weeks with anti-IL-23, anti-IL-24, orIL-22 or an irrelevant control antibody (Con) (as per FIG. 9). HepaticER stress assessed by measurement of spliced-Xbp1 (A) and Hspa5 (Grp78)(B) mRNA by qRT-PCR after 3 weeks treatment. Normalization against thehousekeeping gene Gapdh, and expression as fold difference to theuntreated normal chow control mice.

FIG. 15: Extended IL-22 treatment restores both glucose tolerance andinsulin sensitivity in HFDIO. Mice were fed a high fat diet (HFD) ornormal chow diet (NCD) for 22 weeks and treated for the last 30 dayswith 20 or 100 ng/g IL-22 twice weekly. NCD and HFD control micereceived an irrelevant IgG control antibody. (A) Body weight. (B) Randomfed blood glucose concentrations during treatment. (C-E) Blood glucoseduring a fasted i.p. glucose tolerance test on day 25 (C), and insulintolerance tests on day 16 (D) and 29 (E) of treatment. AUC=area undercurve. (F) Serum total insulin during fasted i.p. glucose tolerance teston day 25. (G) Redistribution of fat in IL-22-treated obese mice.

FIG. 16: Extended IL-22 treatment reduces serum proinsulin and isletproinsulin accumulation in HFDIO. Mice were fed a high fat diet (HFD) ornormal chow diet (NCD) for 22 weeks and treated for the last 30 dayswith 20 or 100 ng/g IL-22 twice weekly. NCD and HFD control micereceived an irrelevant IgG control antibody. (A) Fasted serum proinsulinat the end of treatment. (B) Co-staining of total insulin (green) andproinsulin (magenta) analyzed by confocal-microscopy. Individual singlecolor images are shown at the top, and dual color images are shown atthe bottom. Color reproductions of FIG. 16 are available upon request.

FIG. 17: Extended IL-22 treatment normalizes islet insulin secretion inHFDIO. Mice were fed a high fat diet (HFD) or normal chow diet (NCD) for22 weeks and treated for the last 30 days with 20 or 100 ng/g IL-22twice weekly. NCD and HFD control mice received an irrelevant IgGcontrol antibody. (A, B) Total insulin (A) and proinsulin (B) secretionfrom islets from NCD and HFD control and treated mice on day 30 culturedovernight in 2.8 mM glucose, and cultured consecutively for 30 min eachwith 2.8 mM glucose, 20 mM glucose, and 20 mM glucose+100 nM GLP-1. (C)Uptake of fluorescent deoxy-glucose (6-NBDG) by 3T3 adipocytes inresponse to 2 ng/mL of recombinant insulin (rIns) or insulin derivedfrom the glucose plus GLP-1 stimulation of islets shown in (B).

FIG. 18: IL-22 treatment modulates oxidative stress genes and reduces ERstress and inflammation in pancreatic islets. mRNA expression ofoxidative stress (A), ER stress (B), and cytokine and chemokine (C)genes in pancreatic islets from mice suffering from HFDIO (as describedfor FIG. 15). (D) Mip2 mRNA expression in pancreatic islets from mice ona high fat diet (HFD) or normal chow diet (NCD) for 20 weeks culturedfor 24 h in 2.8 mM glucose±50 ng/mL IL-22.

FIG. 19: Expression of IL-22R1 in human tissues. GTEx public domain data(http://www.broadinstitute.org/gtex/) showing human IL22RA1 mRNAexpression across a range of tissues, with highest expression in thepancreas (note log₁₀ scale).

FIG. 20: Survival, viral titer and histological lung injury andinflammation scores in mice infected with pneumovirus (PVM) with andwithout IL-22 administration. Neonatal C57BL/6 mice were treated witheither recombinant mouse IL-22 (rmlL-22, 20 ng/g i.p. every 3 days), orand irrelevant IgG antibody control (12.5 μg/mouse i.p. every six days)prior to infection with 100 plaque forming units (PFU) of pneumovirus ofmice (PVM) or mock infection (naïve) at 7 days of age. Mice wereinfected intranasally, and sacrificed 1, 5 and 6 days post infection(DPI). (A) Mouse survival (7/9 IL-22 treated mice died between day 5 andday 6 and the experiment was ceased on day 6). (B) Abundance of PVMviral RNA measured by qRT-PCR. Expressed as fold change compared to meanof naïve group of respective DPI after correction for expression of thehousekeeping gene β-actin. (C) Blinded lung injury and inflammationscores from hematoxylin and eosin stained sections. Statistics: (A)Kaplan-Meier survival curve (B, C) Box plots show median, IQR and range.N=9 mice/group; one-way ANOVA with Dunnett post-hoc test as shown.

FIG. 21: Representative H&E-stained and PVM-stained lung sections frommice infected with pneumovirus (PVM) with and without IL-22administration. (A) H&E sections showing inflammation in small airways.Arrows highlight immune cells in air spaces and intra-alveolarinterstitium. (B) Rabbit anti-mouse PVM immunohistochemical staining.

FIG. 22: IL-22 protects human pancreatic islets from the development ofoxidative stress. Human pancreatic islets from a single healthy donorwere cultured for 2 hours in the presence of 50 ng/mL of IFN-γ, IL-18,IL-23 or IL-24, or 0.5 mM palmitate, with or without exposure to 50ng/mL IL-22 beginning 30 min prior to the introduction of the stressinducing cytokines or lipids (n=3 per condition). To test whetherendogenous IL-22 affects human islet physiology the islets were treatedfor 2 hours with an anti-IL-22R1 antibody at 10 μg/mL.

FIG. 23: Human and mouse recombinant IL-22 reduce ER stress in murinecells and human cells, respectively. (a) Murine MIN6N8 cells weretransfected with the ERAI-sXBP1 reporter. After 48 h cells were treatedrecombinant mouse (rm) or human (rh) IL-22 (0.12-15.4 nM concentrations)in the presence of 0.5 mM palmitate for 24 h. Data is presented as thefluorescence intensity. n=4 (pooled data from two individualexperiments). (b) Human LS174T cells were treated with increasingconcentrations of recombinant human or mouse IL-22 2 h prior to theintroduction of 0.5 mM palmitate for 30 min. Cells were lysed andintracellular nitrite was measured using the Griess Assay. n=4 (pooleddata from two individual experiments). Con=PBS (black bar/dotted line)and palmitate alone (0.5 mM; red bar). Histograms show mean±s.e.m.*#P<0.05, **##P<0.01, ***###P<0.001 (from post hoc test); * versusuntreated vehicle control, #versus IL-22 from alternate species atcomparable concentration.

FIG. 24: Modulation of oxidative and ER stress in murine MIN6N8 cellsexposed to palmitate, to recombinant mouse and human IL-22, toIL-22-GLP-1R ligand fusion proteins or to IL-22-ScaB1 scFv construct.MIN6N8 cells transfected with the ERAI-sXBP1 reporter were treated with0.5 mM palmitate and recombinant mouse IL-22 (rmlL-22), recombinanthuman IL-22 (rhIL-22) and the IL-22-GLP-1R ligand fusion proteins(MPBS-50, -51 and -52) and IL-22 ScaB1 at 15.4 nM concentrations for 24hours. PBS and 0.5% BSA were used as vehicle controls. Data shows thatrecombinant IL-22 and the biologic drugs do not cause any ER stressalone.

FIG. 25: Protection against oxidative and ER stress in murine MIN6N8cells or human LS174T cells concomitantly exposed to palmitate and mouseor human IL-22 or different IL-22-GLP-1R ligand fusion proteins. (A)MIN6N8 cells were transfected with the ERAI-sXBP1 reporter wereco-treated with 0.5 mM palmitate and increasing concentrations ofrecombinant human IL-22 (hIL-22) and the IL-22-GLP-1R ligand fusionproteins (MPBS-50, -51 and -52) for 24 hours. N=4 (pooled data from twoindividual experiments). Data is presented as a percentage reduction ofER stress. (B) LS174T cells were treated with increasing concentrationsof recombinant mouse IL-22 (mIL-22), recombinant human IL-22 (hIL-22)and the IL-22-GLP-1R ligand fusion proteins (MPBS-50, 51 and 52) for 2 hprior to the introduction of 0.5 mM palmitate for 30 min. Cells weresubsequently lysed and the Griess assay was used to determine thenitrite concentration. N=4 (pooled data from two individualexperiments). Data is presented as a percentage reduction of oxidativestress (nitrite concentration).

FIG. 26: Reduction of ER stress by IL-22-ScaB1. Reduction of ER stressby IL-22-ScaB1. MIN6N8 cells transfected with the ERAI-sXBP1 reporterwere co-treated with 0.5 mM palmitate and increasing concentrations ofrecombinant human IL-22 (hIL-22) and the IL-22-ScaB1 for 24 hours. N=4.Data is presented as a percentage reduction of ER stress.

FIG. 27: Clearance of IL-22 and IL-22 fusion proteins. Mice wereinjected with 256 ng/g of human IL-22 or the molar equivalents ofNN-IL-22-GLP-1R ligand fusion proteins (MPBS-50, -51,-52). Blood sampleswere collected at different time points as shown.

FIG. 28: Assessment of specific targeting to the pancreatic islets ofIL-22-GLP-1R ligand fusion protein. IL-22 receptor expressing tissues(liver, intestine, skin and pancreas) from mice treated with 256 ng/g ofrecombinant human IL-22 or the molar equivalents of IL-22-GLP-1R ligandfusion proteins (MPBS-50, -51, -52) for 30 min were stained with pStat3antibody. The intensity of fluorescence per area was assessed usingimage 3 software in 3-4 fields of view for each tissue in each mouse(n=3). Within each mouse the mean fluorescence density from multiplefields was calculated for each tissue and then the ratio of pancreaticislet staining intensity in each of the other tissues (a; liver, b;intestine, c; skin) was calculated. Results are presented as fold changerelative to that observed with recombinant human IL-22 (rhIL-22).

FIG. 29: Assessment of specific targeting to the pancreatic islets ofIL-22-ScaB1. IL-22 receptor expressing tissues (liver, intestine, skinand pancreas) from mice treated with 15.4 nmoles/g of recombinant humanIL-22 or 0.96, 3.8 or 15.4 nmoles/g of the IL-22-ScaB1 fusion proteinfor 30 min were stained with pStat3 antibody. The intensity offluorescence per area was assessed using image 3 software in 3-4 fieldsof view for each tissue in each mouse (n=3). Within each mouse the meanfluorescence density from multiple fields was calculated for each tissueand then the ratio of pancreatic islet staining intensity in each of theother tissues (a; liver, b; intestine, c; skin) was calculated. Resultsare presented as fold change relative to that observed with recombinanthuman IL-22 (rhIL-22). d shows pStat3 with human IL-22 (15.4 nmoles/g)and IL-22-ScaB1 (15.4 nmoles/g) in the intestine, liver and pancreas.

FIG. 30: Dose-finding efficacy study in mice with high fat diet inducedobesity with IL-22-GLP-1R ligand fusion proteins. Mice were fed a highfat diet (HFD) or normal chow diet (NCD) for 27 weeks. HFD mice weretreated with 0.06-15.4 nmoles/g of recombinant human IL-22 (rhIL-22) orIL-22-GLP-1R ligand fusion proteins (MPBS-50, -51, -52). Glucosetolerance was measured by an intraperitoneal (i.p.) glucose tolerancetest (IPGTT) (fasted mice were given 40% glucose 2 g/kg i.p. and bloodglucose measured over 120 min).

FIG. 31: IL-22-GLP-1R ligand fusion proteins are more efficacious thanrh IL-22 at lower doses. Mice were fed a high fat diet (HFD) or normalchow diet (NCD) for 27 weeks. HFD mice were treated with 0.06-15.4nmoles/g of recombinant human IL-22 (rhIL-22) or IL-22-GLP-1R ligandfusion proteins (MPBS-50, -51, -52). Glucose tolerance was measured byan intraperitoneal (i.p.) glucose tolerance test (IPGTT) (as in FIG.30). Data is presented as the percentage mean reduction in the increaseof the IPGTT area under the curve (AUC) compared to NCD alone (a) andHFD alone (b).

FIG. 32: Assessment of therapeutic efficacy of IL-22-GLP-1R ligandfusion proteins. Mice were fed a high fat diet (HFD) or normal chow diet(NCD) for 27 weeks. HFD mice were treated with 0.06-15.4 nmoles/g ofrecombinant human IL-22 (rhIL-22) or IL-22-GLP-1R ligand fusion proteins(MPBS-50, 51, 52). Glucose tolerance was measured by a fastedintraperitoneal (i.p.) glucose tolerance test (IPGTT) (as in FIG. 30),blood samples were obtained before challenge and following 15, 30, 60and 120 min. Data from the two lowest doses of proteins is presented asthe percentage mean reduction in the increased area under the IPGTTcurve in control PBS-treated HFD mice.

FIG. 33: Body weight changes. Mice were fed a high fat diet (HFD) ornormal chow diet (NCD) for 27 weeks. HFD mice were treated with0.06-15.4 nmoles/g of recombinant human IL-22 (a; rhIL-22) orIL-22-GLP-1R ligand fusion proteins (b; MPBS-50, c; -51, d; -52). Bodyweight changes were recorded on days 0, 5, 10 and 13. Data is presentedas a percentage of starting body weight on day 0.

FIG. 34: Amino acid sequences of IL-22-ScaB1 constructs and componentparts. a) ScaB1 scFv sequence includes the variable heavy (VH) chain ingrey and the black is the variable light or Kappa (VL or VK) chain. TheGlycine-Serine motif between the two domains forms the flexible linker,(G4S)3, which allows the scFv to fold into a functional targetingmoiety. b) Final sequence used as the human IL22 domain. c) Sequenceshowing design of initial fusion construct (His-ScaB1 scFv-IL22human-myc) with flanking tags to aid in detection.

FIG. 35: PAGE gel of purified His-ScaB1 scFv-IL22 human-myc. a)Illustration of SeeBlue Plus2 pre-stained protein standard (Lifetechnologies). b) Lane 1 shows the protein standard, lane 2 is ofreduced protein and lane 3 is of the non-reduced protein.

FIG. 36: Amino acid sequences of IL-22-GLP-1R ligand fusion proteins.Amino acid sequences of MPBS-50, MPBS-51 and MPBS-52 fusion proteins.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

The terms “administration concurrently” or “administering concurrently”or “co-administering” and the like refer to the administration of asingle composition containing two or more actives, or the administrationof each active as separate compositions and/or delivered by separateroutes either contemporaneously or simultaneously or sequentially withina short enough period of time that the effective result is equivalent tothat obtained when all such actives are administered as a singlecomposition. By “simultaneously” is meant that the active agents areadministered at substantially the same time, and desirably together inthe same formulation. By “contemporaneously” it is meant that the activeagents are administered closely in time, e.g., one agent is administeredwithin from about one minute to within about one day before or afteranother. Any contemporaneous time is useful. However, it will often bethe case that when not administered simultaneously, the agents will beadministered within about one minute to within about eight hours andpreferably within less than about one to about four hours. Whenadministered contemporaneously, the agents are suitably administered atthe same site on the subject. The term “same site” includes the exactlocation, but can be within about 0.5 to about 15 centimeters,preferably from within about 0.5 to about 5 centimeters. The term“separately” as used herein means that the agents are administered at aninterval, for example at an interval of about a day to several weeks ormonths. The active agents may be administered in either order. The term“sequentially” as used herein means that the agents are administered insequence, for example at an interval or intervals of minutes, hours,days or weeks. If appropriate the active agents may be administered in aregular repeating cycle.

By “antigen-binding molecule” is meant a molecule that has bindingaffinity for a target antigen. It will be understood that this termextends to immunoglobulins, immunoglobulin fragments andnon-immunoglobulin derived protein frameworks that exhibitantigen-binding activity. Representative antigen-binding molecules thatare useful in the practice of the present invention include polyclonaland monoclonal antibodies as well as their fragments (such as Fab, Fab′,F(ab′)2, Fv), single chain (scFv) and domain antibodies (including, forexample, shark and camelid antibodies), and fusion proteins comprisingan antibody, and any other modified configuration of the immunoglobulinmolecule that comprises an antigen binding/recognition site. An antibodyincludes an antibody of any class, such as IgG, IgA, or IgM (orsub-class thereof), and the antibody need not be of any particularclass.

Depending on the antibody amino acid sequence of the constant region ofits heavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chainconstant regions that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.Antigen-binding molecules also encompass dimeric antibodies, as well asmultivalent forms of antibodies. In some embodiments, theantigen-binding molecules are chimeric antibodies in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(see, for example, U.S. Pat. No. 4,816,567; and Morrison et al., 1984,Proc. Natl. Acad. Sci. USA, 81:6851-6855). Also contemplated, arehumanized antibodies, which are generally produced by transferringcomplementarity determining regions (CDRs) from heavy and light variablechains of a non-human (e.g., rodent, preferably mouse) immunoglobulininto a human variable domain. Typical residues of human antibodies arethen substituted in the framework regions of the non-human counterparts.The use of antibody components derived from humanized antibodiesobviates potential problems associated with the immunogenicity ofnon-human constant regions. General techniques for cloning non-human,particularly murine, immunoglobulin variable domains are described, forexample, by Orlandi et al. (1989, Proc. Natl. Acad. Sci. USA 86: 3833).Techniques for producing humanized monoclonal antibodies are described,for example, by Jones et al. (1986, Nature 321:522), Carter et al.(1992, Proc. Natl. Acad. Sci. USA 89: 4285), Sandhu (1992, Crit. Rev.Biotech. 12: 437), Singer et al. (1993, J. Immun. 150: 2844), Sudhir(ed., Antibody Engineering Protocols, Humana Press, Inc. 1995), Kelley(“Engineering Therapeutic Antibodies,” in Protein Engineering:Principles and Practice Cleland et al. (eds.), pages 399-434 (John Wiley& Sons, Inc. 1996), and by Queen et al., U.S. Pat. No. 5,693,762 (1997).Humanized antibodies include “primatized” antibodies in which theantigen-binding region of the antibody is derived from an antibodyproduced by immunizing macaque monkeys with the antigen of interest.Also contemplated as antigen-binding molecules are humanized antibodies.

The terms “beta-cell,” “β-cell” or “pancreatic β-cell” are usedinterchangeably herein to refer to cells in the pancreatic islets thatare of the lineage of cells that produce insulin in response to glucose.β-cells are found in the islets of Langerhans in the pancreas.

The term “biologically active fragment,” as applied to fragments of areference or full-length polynucleotide or polypeptide sequence, refersto a fragment that has at least about 0.1, 0.5, 1, 2, 5, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 96, 97, 98, 99% of the activity of a reference sequence.Suitably, the biologically active fragment has no less than about 1%,10%, 25% 50% of an activity of the full-length polypeptide from which itis derived. Included within the scope of the present invention arebiologically active fragments of at least about 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160,180, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000nucleotides or residues in length, which comprise or encode an activityof a reference polynucleotide or polypeptide. Representativebiologically active fragments generally participate in an interaction,e.g., an intra-molecular or an inter-molecular interaction. Aninter-molecular interaction can be a specific binding interaction or anenzymatic interaction (e.g., the interaction can be transient and acovalent bond is formed or broken). For instance, biologically activefragments of a full-length IL-22 or IL-10 polypeptide include peptidescomprising amino acid sequences sufficiently similar to or derived fromthe amino acid sequences of a (putative) full-length precursor or matureIL-22 or IL-10 polypeptide. For example, biologically active portions ofa full-length precursor or mature IL-22 or IL-10 polypeptide includepeptides or polypeptides comprising amino acid sequences with sufficientsimilarity or identity to or derived from the amino acid sequence of afull-length precursor or mature IL-22 or IL-10 polypeptide, as forexample set forth in SEQ ID NO: 2, 4, 6, or 8 and comprises at least onedomain or motif capable of inhibiting OER stress. In some embodiments,the biologically active portions comprise at least one domain or motifcapable of activating the IL-22 receptor (IL-22R) or IL-10 receptor(IL-10R).

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. Thus, use of the term “comprising” and the likeindicates that the listed elements are required or mandatory, but thatother elements are optional and may or may not be present. By“consisting of” is meant including, and limited to, whatever follows thephrase “consisting of”. Thus, the phrase “consisting of” indicates thatthe listed elements are required or mandatory, and that no otherelements may be present. By “consisting essentially of” is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements. Thus, thephrase “consisting essentially of” indicates that the listed elementsare required or mandatory, but that other elements are optional and mayor may not be present depending upon whether or not they affect theactivity or action of the listed elements.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, which can be generally sub-classified asfollows:

TABLE 1 AMINO ACID SUB-CLASSIFICATION SUB-CLASSES AMINO ACIDS AcidicAspartic acid, Glutamic acid Basic Noncyclic: Arginine, Lysine; Cyclic:Histidine Charged Aspartic acid, Glutamic acid, Arginine, Lysine,Histidine Small Glycine, Serine, Alanine, Threonine, ProlinePolar/neutral Asparagine, Histidine, Glutamine, Cysteine, Serine,Threonine Polar/large Asparagine, Glutamine Hydrophobic Tyrosine,Valine, Isoleucine, Leucine, Methionine, Phenylalanine, TryptophanAromatic Tryptophan, Tyrosine, Phenylalanine Residues that influenceGlycine and Proline chain orientation

Conservative amino acid substitution also includes groupings based onside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. For example, it is reasonable to expect that replacementof a leucine with an isoleucine or valine, an aspartate with aglutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid will not have a majoreffect on the properties of the resulting variant polypeptide. Whetheran amino acid change results in a functional polypeptide can readily bedetermined by assaying its activity. Conservative substitutions areshown in Table 2 under the heading of exemplary and preferredsubstitutions. Amino acid substitutions falling within the scope of theinvention, are, in general, accomplished by selecting substitutions thatdo not differ significantly in their effect on maintaining (a) thestructure of the peptide backbone in the area of the substitution, (b)the charge or hydrophobicity of the molecule at the target site, or (c)the bulk of the side chain. After the substitutions are introduced, thevariants are screened for biological activity.

TABLE 2 EXEMPLARY AND PREFERRED AMINO ACID SUBSTITUTIONS ORIGINALEXEMPLARY PREFERRED RESIDUE SUBSTITUTIONS SUBSTITUTIONS Ala Val, Leu,Ile Val Arg Lys, Gln, Asn Lys Asn Gln, His, Lys, Arg Gln Asp Glu Glu CysSer Ser Gln Asn, His, Lys, Asn Glu Asp, Lys Asp Gly Pro Pro His Asn,Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleu Leu Leu Norleu,Ile, Val, Met, Ala, Phe Ile Lys Arg, Gln, Asn Arg Met Leu, Ile, Phe LeuPhe Leu, Val, Ile, Ala Leu Pro Gly Gly Ser Thr Thr Thr Ser Ser Trp TyrTyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Leu, Met, Phe, Ala, Norleu Leu

By “corresponds to” or “corresponding to” is meant an amino acidsequence that displays substantial sequence similarity or identity to areference amino acid sequence. In general the amino acid sequence willdisplay at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%or even up to 100% sequence similarity or identity to at least a portionof the reference amino acid sequence

“Diabetic dyslipidemia” or “Type 2 diabetes with dyslipidemia” means acondition characterized by Type 2 diabetes, reduced HDL-C, elevatedserum triglycerides, and elevated small, dense LDL particles.

By “effective amount,” in the context of inhibiting ER stress ortreating or preventing a disease or condition, is meant theadministration of an amount of agent to an individual in need thereof,either in a single dose or as part of a series, that is effective forthat inhibition, treatment or prevention. The effective amount will varydepending upon the health and physical condition of the individual to betreated, the taxonomic group of individual to be treated, theformulation of the composition, the assessment of the medical situation,and other relevant factors. It is expected that the amount will fall ina relatively broad range that can be determined through routine trials.

The term “ER stress” refers to the biochemical state and cellularresponse associated with (e.g., caused by, correlated with, or inducedby) protein misfolding or other disturbances to normal physiology withinthe endoplasmic reticulum. These cellular responses include, but are notlimited to, gene expression, protein translation/expression, and proteindegradation. Various methodologies described herein include steps thatinvolve determining or comparing levels of ER stress signaling. Methodsfor determining levels of ER stress are known in the art. For example,methods for measuring ER stress signaling are described in U.S. Pat.Publication No. 20070202544, the contents of which are incorporatedherein by reference. Example 1 herein also describes exemplary methodsfor measuring level of ER stress. For example, expression levels of ERstress response genes, e.g., expression and splicing of the Xbp-1 mRNAcan be measured.

The term “euglycemia” is defined as the condition in which a subject hasa fasting blood glucose concentration within the normal range, greaterthan 70 mg/dL (3.89 mmol/L) and less than 100 mg/dL (5.6 mmol/L).

By “gene” is meant a unit of inheritance that occupies a specific locuson a chromosome and consists of transcriptional and/or translationalregulatory sequences and/or a coding region and/or non-translatedsequences (i.e., introns, 5′ and 3′ untranslated sequences).

As used herein, the term “hyperfattyacidemia” refers to a conditioncharacterized by elevated fatty acid levels. Exemplary total fatty acidlevels considered abnormally elevated in a human subject is 0.72 mmol/Land above.

The term “hyperlipidemia” refers to the presence of an abnormallyelevated level of lipids in the blood. Hyperlipidemia can appear in atleast three for MS (ES): (1) hypercholesterolemia, i.e., an elevated LDLcholesterol level (120 mg/dL and above); (2) hypertriglyceridemia, i.e.,an elevated triglyceride level; (150 mg/dL and above) and (3) combinedhyperlipidemia, i.e., a combination of hypercholesterolemia andhypertriglyceridemia.

The term “hypertension” as used herein includes essential, or primary,hypertension wherein the cause is not known or where hypertension is dueto greater than one cause, such as changes in both the heart and bloodvessels; and secondary hypertension wherein the cause is known. Causesof secondary hypertension include, but are not limited to obesity;kidney disease; hormonal disorders; use of certain drugs, such as oralcontraceptives, corticosteroids, cyclosporine, and the like. The term“hypertension” encompasses high blood pressure, in which both thesystolic and diastolic pressure levels are elevated 140 mmHg/90 mmHg),and isolated systolic hypertension, in which only the systolic pressureis elevated to greater than or equal to 140 mm Hg, while the diastolicpressure is less than 90 mm Hg. Normal blood pressure may be defined asless than 120 mmHg systolic and less than 80 mmHg diastolic.

As used herein, the term “hypertriglyceridemia” refers to a conditioncharacterized by elevated triglyceride levels. Exemplary totaltriglyceride levels considered abnormally elevated in a human subject is150 mg/dL and above.

The term “hyperinsulinemia” is defined as the condition in which asubject with insulin resistance, with or without euglycemia, has fastingor postprandial serum or plasma insulin concentration elevated abovethat of normal, lean individuals without insulin resistance, having awaist-to-hip ratio <1.0 (for men) or <0.8 (for women).

The term “IL-10 polypeptide,” as used herein encompasses, withoutlimitation, polypeptides having an amino acid sequence that shares atleast 70% (and at least 71% to at least 99% and all integer percentagesin between) sequence identity or similarity with the sequence set forthin any one of SEQ ID NO: 4. It further encompasses natural allelicvariation of IL-10 polypeptides that may exist and occur from oneorganism to another. Also, degree and location of glycosylation or otherpost-translation modifications may vary depending on the chosen host andthe nature of the host's cellular environment. The term “IL-10polypeptide” is also intended to encompass IL-10 polypeptides in theirprecursor form, as well as those that have been processed to yield theirrespective bioactive forms. It further encompasses IL-10 polypeptidesthat have either been chemically modified relative to a reference ornaturally-occurring IL-10 polypeptide and/or contain one or more aminoacid sequence alterations relative to a reference or naturally-occurringIL-10 polypeptide and/or contain truncated amino acid sequences relativeto a reference or naturally-occurring full-length or precursor IL-10polypeptide. These truncated sequences are typically biologically activefragments of a reference or naturally occurring full-length or precursorIL-10 polypeptide. IL-10 polypeptides may exhibit different propertiesrelative to a reference or naturally-occurring IL-10 polypeptide,including stability and an altered specific activity selected fromstimulating or otherwise inducing apoptosis of an adipose cell ortissue; reducing fasting hyperinsulinemia, reducing glucose levels aftera hyperglycemic stimulus; reducing hyperinsulinemia after ahyperglycemic stimulus, enhancing peripheral response to insulin;reducing increased adiposity in response to high fat diet, improvingmitochondrial fatty acid oxidative capacity of muscle tissue, reducingcirculating levels of IL-6, and the like. The term “IL-10 polypeptide”also encompasses proteinaceous molecules with a slightly modified aminoacid sequence, for instance, polypeptides having a modified N-terminalend including N-terminal amino acid deletions or additions, and/orpolypeptides that have been chemically modified relative to a referenceor naturally-occurring IL-10 polypeptide. IL-10 polypeptides alsoencompass proteinaceous molecules exhibiting substantially the same orbetter bioactivity than a reference or naturally occurring IL-10polypeptide, or, alternatively, exhibiting substantially modified orreduced bioactivity relative to a reference or naturally-occurring IL-10polypeptide. They also include, without limitation, polypeptides havingan amino acid sequence that differs from the sequence of a reference ornaturally-occurring IL-10 polypeptide by insertion, deletion, orsubstitution of one or more amino acids and in illustrative examples,encompass proteinaceous molecules that exhibit at least about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, and 130% of thespecific activity of a reference or naturally occurring IL-10polypeptide that has been produced in the same cell. IL-10 polypeptideshaving substantially the same or improved biological activity relativeto a reference or naturally-occurring IL-10 polypeptide, encompassmolecules that exhibit at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 110%, 120%, and 130% of the specific biological activityof the reference or naturally-occurring IL-10 polypeptide that has beenproduced in the same cell type.

The term “IL-22 polypeptide,” as used herein encompasses, withoutlimitation, polypeptides having an amino acid sequence that shares atleast 70% (and at least 71% to at least 99% and all integer percentagesin between) sequence identity or similarity with the sequence set forthin any one of SEQ ID NO: 1. It further encompasses natural allelicvariation of IL-22 polypeptides that may exist and occur from oneorganism to another. Also, degree and location of glycosylation or otherpost-translation modifications may vary depending on the chosen host andthe nature of the host's cellular environment. The term “IL-22polypeptide” is also intended to encompass IL-22 polypeptides in theirprecursor form, as well as those that have been processed to yield theirrespective bioactive forms. It further encompasses IL-22 polypeptidesthat have either been chemically modified relative to a reference ornaturally-occurring IL-22 polypeptide and/or contain one or more aminoacid sequence alterations relative to a reference or naturally-occurringIL-22 polypeptide and/or contain truncated amino acid sequences relativeto a reference or naturally-occurring full-length or precursor IL-22polypeptide. These truncated sequences are typically biologically activefragments of a reference or naturally occurring full-length or precursorIL-22 polypeptide. IL-22 polypeptides may exhibit different propertiesrelative to a reference or naturally-occurring IL-22 polypeptide,including stability and an altered specific activity selected fromstimulating or otherwise inducing apoptosis of an adipose cell ortissue; reducing fasting hyperinsulinemia, reducing glucose levels aftera hyperglycemic stimulus; reducing hyperinsulinemia after ahyperglycemic stimulus, enhancing peripheral response to insulin;reducing increased adiposity in response to high fat diet, improvingmitochondrial fatty acid oxidative capacity of muscle tissue, reducingcirculating levels of IL-6, and the like. The term “IL-22 polypeptide”also encompasses proteinaceous molecules with a slightly modified aminoacid sequence, for instance, polypeptides having a modified N-terminalend including N-terminal amino acid deletions or additions, and/orpolypeptides that have been chemically modified relative to a referenceor naturally-occurring IL-22 polypeptide. IL-22 polypeptides alsoencompass proteinaceous molecules exhibiting substantially the same orbetter bioactivity than a reference or naturally occurring IL-22polypeptide, or, alternatively, exhibiting substantially modified orreduced bioactivity relative to a reference or naturally-occurring IL-22polypeptide. They also include, without limitation, polypeptides havingan amino acid sequence that differs from the sequence of a reference ornaturally-occurring IL-22 polypeptide by insertion, deletion, orsubstitution of one or more amino acids and in illustrative examples,encompass proteinaceous molecules that exhibit at least about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, and 130% of thespecific activity of a reference or naturally occurring IL-22polypeptide that has been produced in the same cell. IL-22 polypeptideshaving substantially the same or improved biological activity relativeto a reference or naturally-occurring IL-22 polypeptide, encompassmolecules that exhibit at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 110%, 120%, and 130% of the specific biological activityof the reference or naturally-occurring IL-22 polypeptide that has beenproduced in the same cell type.

The term “insulin resistance” refers to a state in which a normal amountof insulin produces a subnormal biologic response relative to thebiological response in a subject that does not have insulin resistance.

“Insulin sensitivity” means the ability of cells to take up glucose inresponse to insulin action.

By “linker” is meant a molecule or group of molecules (such as a monomeror polymer) that connects two molecules and often serves to place thetwo molecules in a desirable configuration.

“Metabolic syndrome” means a condition characterized by a clustering oflipid and non-lipid risk factors of metabolic origin. In certainembodiments, metabolic syndrome is identified by the presence of any 3of the following factors: waist circumference of greater than 102 cm inmen or greater than 88 cm in women; serum triglyceride of at least 150mg/dL; HDL-C less than 40 mg/dL in men or less than 50 mg/dL in women;blood pressure of at least 130/85 mmHg; and fasting glucose of at least110 mg/dL. These determinants can be readily measured in clinicalpractice (JAMA, 2001, 285: 2486-2497).

By “modulating” is meant increasing or decreasing, either directly orindirectly, the level or functional activity of a target molecule. Forexample, an agent may indirectly modulate the level/activity byinteracting with a molecule other than the target molecule. In thisregard, indirect modulation of a gene encoding a target polypeptideincludes within its scope modulation of the expression of a firstnucleic acid molecule, wherein an expression product of the firstnucleic acid molecule modulates the expression of a nucleic acidmolecule encoding the target polypeptide.

“Non-alcoholic fatty liver disease (NAFLD)” means acondition-characterized accumulation of fat in the liver in subjects whoconsume little to no alcohol. In certain embodiments, NAFLD is relatedto insulin resistance and the metabolic syndrome.

“Nonalcoholic steatohepatitis (NASH)” means a condition characterized byaccumulation of fat in the liver, combined with inflammation ±scarringin the liver. In certain embodiments NASH results from a worseningprogression of NAFLD.

The term “obesity” as used herein includes conditions where there is anincrease in body fat beyond the physical requirement as a result ofexcess accumulation of adipose tissue in the body. Generally, the term“obesity” refers to an excessively high amount of body fat or adiposetissue in relation to lean body mass. The amount of body fat (oradiposity) includes both the distribution of fat throughout the body andthe size of the adipose tissue deposits. Body fat distribution can beestimated by skin-fold measures, waist-to-hip circumference ratios, ortechniques such as ultrasound, computed tomography, or magneticresonance imaging. According to the Center for Disease Control andPrevention, individuals with a body mass index (BMI) of 30 or more areconsidered obese. The term obesity includes, but is not limited to, thefollowing conditions: adult-onset obesity; alimentary obesity;endogenous or metabolic obesity; endocrine obesity; familial obesity;hyperinsulinar obesity; hyperplastic-hypertrophic obesity; hypogonadalobesity; hypothyroid obesity; lifelong obesity; morbid obesity andexogenous obesity. Similarly, the term “diet-induced obesity” (DIO), isa model created to study obesity and its co-morbidities such as type 2diabetes, hypertension, hypercholesterolemia, and atherosclerosis. Inthis model, an animal (mouse, rat, dog, or non-human primate) is fed ahigh fat and/or high sugar diet for a number of weeks. As a result, itbecomes obese, and usually hyperglycemic, and develops impaired glucosetolerance.

By “obtained from” is meant that a sample such as, for example, apolynucleotide extract or polypeptide extract is isolated from, orderived from, a particular source.

The term “oxidative stress” as used herein refers to a cell alterationcharacterized by an excessive production of reactive oxygen species(ROS) and/or reactive nitrogen species (RNS) and a loss of efficacy ofantioxidative defenses leading to pathological states in the cell andcausing cell or tissue damage. Examples of such damage include but arenot limited to oxidation of lipoproteins; membrane phospholipids; lipidperoxidation; protein damage, including cleavage of amino acid bonds andoxidation of functional groups; nucleic acid strand breaks; nucleic acidbase modifications leading to point mutations; inhibition of RNA andprotein synthesis; protein cross-linking; impaired maintenance ofmembrane ion gradients; and depletion of cellular levels of ATP, leadingto cellular dysfunction and eventually to disease. The oxidant(oxidizing reagent) can be endogenous or exogenous.

The terms “patient,” “subject,” “host” or “individual” usedinterchangeably herein, refer to any subject, particularly a vertebratesubject, and even more particularly a mammalian subject, for whomtherapy or prophylaxis is desired. Suitable vertebrate animals that fallwithin the scope of the invention include, but are not restricted to,any member of the subphylum Chordata including primates (e.g., humans,monkeys and apes, and includes species of monkeys such from the genusMacaca (e.g., cynomologus monkeys such as Macaca fascicularis, and/orrhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus), as well asmarmosets (species from the genus Callithrix), squirrel monkeys (speciesfrom the genus Saimiri) and tamarins (species from the genus Saguinus),as well as species of apes such as chimpanzees (Pan troglodytes)),rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits,hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g.,goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g.,dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks,geese, companion birds such as canaries, budgerigars etc.), marinemammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizardsetc.), and fish. A preferred subject is a human in need of reducing OERstress in a cell, for example a β-cell (e.g., a human having or at riskof a metabolic disorder). However, it will be understood that theaforementioned terms do not imply that symptoms are present.

“Pre-diabetes” means a condition in which a subject's blood glucoselevels are higher than in a subject with normal blood glucose levels andlower but not high enough for a diagnosis of diabetes.

By “pharmaceutically acceptable carrier” is meant a solid or liquidfiller, diluent or encapsulating substance that can be safely used intopical or systemic administration to an animal, preferably a mammal,including humans. Representative pharmaceutically acceptable carriersinclude any and all solvents, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp.1289-1329, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient(s), itsuse in the pharmaceutical compositions is contemplated.

The term “polynucleotide” or “nucleic acid” as used herein designatesmRNA, RNA, cRNA, cDNA or DNA. The term typically refers to polymericform of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide. The term includes single and double stranded forms of DNA.

The terms “polynucleotide variant” and “variant” and the like refer topolynucleotides displaying substantial sequence identity with areference polynucleotide sequence or polynucleotides that hybridize witha reference sequence under stringent conditions that are definedhereinafter. These terms also encompass polynucleotides that aredistinguished from a reference polynucleotide by the addition, deletionor substitution of at least one nucleotide. Accordingly, the terms“polynucleotide variant” and “variant” include polynucleotides in whichone or more nucleotides have been added or deleted, or replaced withdifferent nucleotides. In this regard, it is well understood in the artthat certain alterations inclusive of mutations, additions, deletionsand substitutions can be made to a reference polynucleotide whereby thealtered polynucleotide retains the biological function or activity ofthe reference polynucleotide. The terms “polynucleotide variant” and“variant” also include naturally occurring allelic variants.

“Polypeptide,” “peptide,” “protein” and “proteinaceous molecule” areused interchangeably herein to refer to molecules comprising orconsisting of a polymer of amino acid residues and to variants andsynthetic analogues of the same. Thus, these terms apply to amino acidpolymers in which one or more amino acid residues are syntheticnon-naturally occurring amino acids, such as a chemical analogue of acorresponding naturally occurring amino acid, as well as tonaturally-occurring amino acid polymers.

The terms “peptide variant” and “polypeptide variant” and the like referto peptides and polypeptides that are distinguished from a referencepeptide or polypeptide by the addition, deletion or substitution of atleast one amino acid residue. In certain embodiments, a peptide orpolypeptide variant is distinguished from a reference peptide orpolypeptide by one or more substitutions, which may be conservative ornon-conservative. In certain embodiments, the peptide or polypeptidevariant comprises conservative substitutions and, in this regard, it iswell understood in the art that some amino acids may be changed toothers with broadly similar properties without changing the nature ofthe activity of the peptide or polypeptide. Peptide and polypeptidevariants also encompass peptides and polypeptides in which one or moreamino acids have been added or deleted, or replaced with different aminoacid residues.

By “recombinant polypeptide” is meant a polypeptide made usingrecombinant techniques, i.e., through the expression of a recombinantpolynucleotide.

The term “sequence identity” as used herein refers to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. The present invention contemplates the use in the methods andsystems of the present invention of full-length IL-22 polypeptides aswell as their biologically active fragments. Typically, biologicallyactive fragments of a full-length IL-22 polypeptide may participate inan interaction, for example, an intra-molecular or an inter-molecularinteraction.

“Similarity” refers to the percentage number of amino acids that areidentical or constitute conservative substitutions as defined in Tables1 and 2 supra. Similarity may be determined using sequence comparisonprograms such as GAP (Deveraux et al. 1984, Nucleic Acids Research 12:387-395). In this way, sequences of a similar or substantially differentlength to those cited herein might be compared by insertion of gaps intothe alignment, such gaps being determined, for example, by thecomparison algorithm used by GAP.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence,”“comparison window”, “sequence identity,” “percentage of sequenceidentity” and “substantial identity”. A “reference sequence” is at least12 but frequently 15 to 18 and often at least 25 monomer units,inclusive of nucleotides and amino acid residues, in length. Because twopolynucleotides may each comprise (1) a sequence (i.e., only a portionof the complete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window” refers to aconceptual segment of at least 6 contiguous positions, usually about 50to about 100, more usually about 100 to about 150 in which a sequence iscompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. The comparisonwindow may comprise additions or deletions (i.e., gaps) of about 20% orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by computerized implementations of algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) orby inspection and the best alignment (i.e., resulting in the highestpercentage homology over the comparison window) generated by any of thevarious methods selected. Reference also may be made to the BLAST familyof programs as for example disclosed by Altschul et al., 1997, Nucl.Acids Res. 25:3389. A detailed discussion of sequence analysis can befound in Unit 19.3 of Ausubel et al., “Current Protocols in MolecularBiology”, John Wiley & Sons Inc, 1994-1998, Chapter 15.

“Stringency” as used herein, refers to the temperature and ionicstrength conditions, and presence or absence of certain organicsolvents, during hybridization and washing procedures. The higher thestringency, the higher the degree of complementarity will be betweenimmobilized target nucleotide sequences and probe nucleotide sequencesthat remain hybridized to the target after washing. Stringencyconditions include low, medium, high and very high stringencyconditions, which describe certain conditions for hybridization andwashing. Guidance for performing hybridization reactions can be found inAusubel et al., (1998, supra), Sections 6.3.1-6.3.6. Aqueous andnon-aqueous methods are described in that reference and either can beused. Reference herein to low stringency conditions include andencompass from at least about 1% v/v to at least about 15% v/v formamideand from at least about 1 M to at least about 2 M salt for hybridizationat 42° C., and at least about 1 M to at least about 2 M salt for washingat 42° C. Low stringency conditions also may include 1% Bovine SerumAlbumin (BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS forhybridization at 65° C., and (i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mMEDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS for washing at room temperature. Oneembodiment of low stringency conditions includes hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by twowashes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of thewashes can be increased to 55° C. for low stringency conditions). Mediumstringency conditions include and encompass from at least about 16% v/vto at least about 30% v/v formamide and from at least about 0.5 M to atleast about 0.9 M salt for hybridization at 42° C., and at least about0.1 M to at least about 0.2 M salt for washing at 55° C. Mediumstringency conditions also may include 1% Bovine Serum Albumin (BSA), 1mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C., and(i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2),5% SDS for washing at 60-65° C. One embodiment of medium stringencyconditions includes hybridizing in 6×SSC at about 45° C., followed byone or more washes in 0.2×SSC, 0.1% SDS at 60° C. High stringencyconditions include and encompass from at least about 31% v/v to at leastabout 50% v/v formamide and from about 0.01 M to about 0.15 M salt forhybridization at 42° C., and about 0.01 M to about 0.02 M salt forwashing at 55° C. High stringency conditions also may include 1% BSA, 1mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C., and(i) 0.2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH7.2), 1% SDS for washing at a temperature in excess of 65° C. Oneembodiment of high stringency conditions includes hybridizing in 6×SSCat about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at65° C. In certain embodiments, a peptide or polypeptide is encoded by apolynucleotide that hybridizes to a reference nucleotide sequence undervery high stringency conditions. One embodiment of very high stringencyconditions includes hybridizing 0.5 M sodium phosphate, 7% SDS at 65°C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Otherstringency conditions are well known in the art and a skilled addresseewill recognize that various factors can be manipulated to optimize thespecificity of the hybridization. Optimization of the stringency of thefinal washes can serve to ensure a high degree of hybridization. Fordetailed examples, see Ausubel et al., supra at pages 2.10.1 to 2.10.16and Sambrook et al. (1989, supra) at sections 1.101 to 1.104. Whilestringent washes are typically carried out at temperatures from about42° C. to 68° C., one skilled in the art will appreciate that othertemperatures may be suitable for stringent conditions. Maximumhybridization rate typically occurs at about 20° C. to 25° C. below theTm for formation of a DNA-DNA hybrid. It is well known in the art thatthe Tm is the melting temperature, or temperature at which twocomplementary polynucleotide sequences dissociate. Methods forestimating Tm are well known in the art (see Ausubel et al., supra atpage 2.10.8). In general, the Tm of a perfectly matched duplex of DNAmay be predicted as an approximation by the formula:

T_(m)=81.5+16.6(log₁₀ M)+0.41(% G+C)˜0.63(% formamide)−(600/length)

wherein: M is the concentration of Na⁺, preferably in the range of 0.01molar to 0.4 molar; % G+C is the sum of guanosine and cytosine bases asa percentage of the total number of bases, within the range between 30%and 75% G+C; % formamide is the percent formamide concentration byvolume; length is the number of base pairs in the DNA duplex. The Tm ofa duplex DNA decreases by approximately 1° C. with every increase of 1%in the number of randomly mismatched base pairs. Washing is generallycarried out at T_(m)−15° C. for high stringency, or T_(m)−30° C. formoderate stringency. In one example of a hybridization procedure, amembrane (e.g., a nitrocellulose membrane or a nylon membrane)containing immobilized DNA is hybridized overnight at 42° C. in ahybridization buffer (50% deionized formamide, 5×SSC, 5×Denhardt'ssolution (0.1% ficoll, 0.1% polyvinylpyrrolidone and 0.1% bovine serumalbumin), 0.1% SDS and 200 mg/mL denatured salmon sperm DNA) containinglabeled probe. The membrane is then subjected to two sequential mediumstringency washes (i.e., 2×SSC, 0.1% SDS for 15 min at 45° C., followedby 2×SSC, 0.1% SDS for 15 min at 50° C.), followed by two sequentialhigher stringency washes (i.e., 0.2×SSC, 0.1% SDS for 12 min at 55° C.followed by 0.2×SSC and 0.1% SDS solution for 12 min at 65-68° C.

“Steatosis” means a condition characterized by the excessiveaccumulation of triglycerides in hepatocytes.

“Steatohepatitis” means steatosis with inflammation.

The term “sulfonylurea compound” and grammatical variations thereof,includes the stereoisomers of the compound, pharmaceutically acceptablesalts of the compound, prodrugs of the compound, and pharmaceuticallyacceptable salts of the prodrugs.

As used herein, the term “therapeutic agent” refers to an agentcomprising the specified elements, as well as any agent that results,directly or indirectly, from the combination of the specified elements.

As used herein, the terms “treatment”, “treating”, and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, i.e., causing regression of the disease.

The term “type 1 diabetes” is defined as the condition in which asubject has, in the presence of autoimmunity towards the pancreaticβ-cell or insulin, a fasting blood glucose or serum glucoseconcentration greater than 125 mg/dL (6.94 mmol/L).

The presence of autoimmunity towards the pancreatic β-cell may beobserved by detection of circulating islet cell autoantibodies [“type IAdiabetes mellitus”], i.e., at least one of: GAD65 [glutamic aciddecarboxylase-65], ICA [islet-cell cytoplasm], IA-2 [intracytoplasmicdomain of the tyrosine phosphatase-like protein IA-2], ZnT8[zinc-transporter-8] or anti-insulin; or other signs of autoimmunitywithout the presence of typical circulating autoantibodies [type IBdiabetes], i.e., as detected through pancreatic biopsy or imaging).Typically a genetic predisposition is present (e.g. HLA, INS VNTR andPTPN22), but this is not always the case.

“Type 2 diabetes” or “non-insulin dependent diabetes mellitus” refers toan insulin-related disorder in which there is a relative disparitybetween endogenous insulin production and insulin requirements, leadingto elevated hepatic glucose production, elevated blood glucose levels,inappropriate insulin secretion, and peripheral insulin resistance. Type2 diabetes has been regarded as a relatively distinct disease entity,but type 2 diabetes is often a manifestation of a much broaderunderlying disorder (Zimmet et al., 2001 Nature 414: 782-787), which mayinclude metabolic syndrome (syndrome X), diabetes (e.g., type 2diabetes, type 2 diabetes, gestational diabetes, autoimmune diabetes),hyperinsulinemia, hyperglycemia, impaired glucose tolerance (IGT),hypoglycemia, β-cell failure, insulin resistance, dyslipidemias,atheroma, insulinoma, hypertension, hypercoagulability,microalbuminuria, and obesity and other adiposity-related conditionssuch as visceral obesity, central fat, obesity-related type 2 diabetes,obesity-related atherosclerosis, heart disease, obesity-related insulinresistance, obesity-related hypertension, microangiopathic lesionsresulting from obesity-related type 2 diabetes, ocular lesions caused bymicroangiopathy in obese individuals with obesity-related type Udiabetes, and renal lesions caused by microangiopathy in obeseindividuals with obesity-related type 2 diabetes.

The terms “wild-type” and “naturally occurring” are used interchangeablyto refer to a gene or gene product that has the characteristics of thatgene or gene product when isolated from a naturally occurring source. Awild type gene or gene product (e.g., a polypeptide) is that which ismost frequently observed in a population and is thus arbitrarilydesigned the “normal” or “wild-type” form of the gene.

As used herein, underscoring or italicizing the name of a gene shallindicate the gene, in contrast to its protein product, which isindicated by the name of the gene in the absence of any underscoring oritalicizing. For example, “IL-22” shall mean the IL-22 gene or IL-22polynucleotides, whereas “IL-22” shall indicate the protein product orproducts generated from transcription and translation and alternativesplicing of the “IL-22” gene.

Each embodiment described herein is to be applied mutatis mutandis toeach and every embodiment unless specifically stated otherwise.

2. OER Stress Inhibitors

2.1 Cytokines that Inhibit or Reduce OER Stress

The present invention provides therapeutic agents that inhibit, reduceor normalize OER stress in a β-cell, and is based in part on thedetermination that some cytokines produced in the local environment inthe pancreatic islets, such as IL-23 and IL-24 promote or induce β-cellOER stress, whilst others such as IL-22 actively inhibit or reduce it.Furthermore, in obesity high concentrations of lipids and glucose, andhigh rates of insulin biosynthesis required due to insulin resistance,contribute to OER stress in β-cells. Thus, the present inventorsconsider that β-cell OER stress inhibitors will be useful in thetreatment of diseases associated with aberrant β-cell ER stress,including metabolic disorders such as diabetes.

The present invention contemplates the use of any molecule that inhibitsor reduces OER stress in a β-cell. In accordance with the presentinvention, the OER stress inhibitor is suitably selected from an IL-22polypeptide, an IL-10 polypeptide or an antagonist of any one of IL-23,IL-24, IL-33, IL-1β, MIP-2α, IL-17A, IFN-γ or IFN-β.

In preferred embodiments, the OER stress inhibitor is an IL-22polypeptide. The present inventors have discovered that IL-22 is apowerful endogenous paracrine suppressor of OER stress in pancreaticislets, and that in obesity-induced hyperglycemia IL-22 therapy restoresglucose control by attenuating defects in pancreatic insulinbiosynthesis and secretion. IL-22 reduces OER stress in pancreaticβ-cells. Notably, IL-22 reduces stress induced by lipids, inflammatorycytokines or environmental ROS, e.g., via STAT1- and STAT3-mediatedupregulation of anti-oxidant genes and suppression of oxidativestress-inducing genes.

Accordingly, in some embodiments the OER stress inhibitor is an IL-22polypeptide comprising, consisting or consisting essentially of an aminoacid sequence selected from:

(a) an amino acid sequence selected from:MAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI [SEQ ID NO:2], which corresponds to the amino acid sequence of the precursor formof IL-22, orAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI [SEQ ID NO: 4], which corresponds to the amino acidsequence of the mature form of IL-22; or

(b) an amino acid sequence that shares at least 70% (and at least 71% toat least 99% and all integer percentages in between) sequence similarityor sequence identity with the sequence set forth in any one of SEQ IDNO: 2 or 4; or

(c) an amino acid sequence that is encoded by a nucleotide sequenceselected from:atggccgccctgcagaaatctgtgagctctttccttatggggaccctggccaccagctgcctccttctcttggccctcttggtacagggaggagcagctgcgcccatcagctcccactgcaggcttgacaagtccaacttccagcagccctatatcaccaaccgcaccttcatgctggctaaggaggctagcttggctgataacaacacagacgttcgtctcattggggagaaactgttccacggagtcagtatgagtgagcgctgctatctgatgaagcaggtgctgaacttcacccttgaagaagtgctgttccctcaatctgataggttccagccttatatgcaggaggtggtgcccttcctggccaggctcagcaacaggctaagcacatgtcatattgaaggtgatgacctgcatatccagaggaatgtgcaaaagctgaaggacacagtgaaaaagcttggagagagtggagagatcaaagcaattggagaactggatttgctgtttatgtctctgagaaatgcctgcatt [SEQ ID NO: 1],which corresponds to the coding sequence for the amino acid sequence setforth in SEQ ID NO: 2, or

gcgcccatcagctcccactgcaggcttgacaagtccaacttccagcagccctatatcaccaaccgcaccttcatgctggctaaggaggctagcttggctgataacaacacagacgttcgtctcattggggagaaactgttccacggagtcagtatgagtgagcgctgctatctgatgaagcaggtgctgaacttcacccttgaagaagtgctgttccctcaatctgataggttccagccttatatgcaggaggtggtgcccttcctggccaggctcagcaacaggctaagcacatgtcatattgaaggtgatgacctgcatatccagaggaatgtgcaaaagctgaaggacacagtgaaaaagcttggagagagtggagagatcaaagcaattggagaactggatttgctgtttatgtctctgagaaatgcctgcatt [SEQ ID NO: 3], whichcorresponds to the coding sequence for the amino acid sequence set forthin SEQ ID NO: 4;

(d) an amino acid sequence that is encoded by a nucleotide sequence thatshares at least 70% (and at least 71% to at least 99% and all integerpercentages in between) sequence identity with the sequence set forth inSEQ ID NO: 1 or 3, or a complement thereof; or

(e) an amino acid sequence that is encoded by a nucleotide sequence thathybridizes under low, medium or high stringency conditions to thesequence set forth in any one of SEQ ID NO: 1 or 3, or a complementthereof,

wherein the amino acid sequence of (a), (b), (c), (d) or (e) has any oneor more activities selected from: improving pancreatic β-cell ERfunction, improving insulin biosynthesis, increasing glucose tolerance,modulating expression of oxidative stress regulatory genes, reducingstress induced by lipids, glucose, inflammatory cytokines orenvironmental ROS, e.g., via STAT1- and STAT3-mediated upregulation ofanti-oxidant genes and suppression of oxidative stress-inducing genes,reducing ER stress, promoting secretion of high quality efficaciousinsulin, restoring glucose homeostasis or enhancing peripheral insulinsensitivity.

In other embodiments, the OER stress inhibitor is an IL-10 polypeptidecomprising, consisting or consisting essentially of an amino acidsequence selected from:

(a) an amino acid sequence selected from:MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN [SEQ ID NO: 6], whichcorresponds to the amino acid sequence of the precursor form of IL-10,or

SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN [SEQ ID NO:8], whichcorresponds to the amino acid sequence of the mature form of IL-10.

(b) an amino acid sequence that shares at least 70% (and at least 71% toat least 99% and all integer percentages in between) sequence similarityor sequence identity with the sequence set forth in any one of SEQ IDNO: 6 or 8; or

(c) an amino acid sequence that is encoded by a nucleotide sequenceselected from:atgcacagctcagcactgctctgttgcctggtcctcctgactggggtgagggccagcccaggccagggcacccagtctgagaacagctgcacccacttcccaggcaacctgcctaacatgcttcgagatctccgagatgccttcagcagagtgaagactttctttcaaatgaaggatcagctggacaacttgttgttaaaggagtccttgctggaggactttaagggttacctgggttgccaagccttgtctgagatgatccagttttacctggaggaggtgatgccccaagctgagaaccaagacccagacatcaaggcgcatgtgaactccctgggggagaacctgaagaccctcaggctgaggctacggcgctgtcatcgatttcttccctgtgaaaacaagagcaaggccgtggagcaggtgaagaatgcctttaataagctccaagagaaaggcatctacaaagccatgagtgagtttgacatcttcatcaactacatagaagcctacatgacaatgaagatacgaaac [SEQ ID NO: 5],which corresponds to the coding sequence for the amino acid sequence setforth in SEQ ID NO: 6, or

agcccaggccagggcacccagtctgagaacagctgcacccacttcccaggcaacctgcctaacatgcttcgagatctccgagatgccttcagcagagtgaagactttctttcaaatgaaggatcagctggacaacttgttgttaaaggagtccttgctggaggactttaagggttacctgggttgccaagccttgtctgagatgatccagttttacctggaggaggtgatgccccaagctgagaaccaagacccagacatcaaggcgcatgtgaactccctgggggagaacctgaagaccctcaggctgaggctacggcgctgtcatcgatttcttccctgtgaaaacaagagcaaggccgtggagcaggtgaagaatgcctttaataagctccaagagaaaggcatctacaaagccatgagtgagtttgacatcttcatcaactacatagaagcctacatgacaatgaagatacgaaac [SEQ ID NO: 7], which corresponds to the coding sequence for theamino acid sequence set forth in SEQ ID NO: 8;

(d) an amino acid sequence that is encoded by a nucleotide sequence thatshares at least 70% (and at least 71% to at least 99% and all integerpercentages in between) sequence identity with the sequence set forth inSEQ ID NO: 5 or 7, or a complement thereof; or

(e) an amino acid sequence that is encoded by a nucleotide sequence thathybridizes under low, medium or high stringency conditions to thesequence set forth in any one of SEQ ID NO: 5 or 7, or a complementthereof,

wherein the amino acid sequence of (a), (b), (c), (d) or (e) has any oneor more activities selected from: improving pancreatic β-cell ERfunction, improving insulin biosynthesis, increasing glucose tolerance,modulating expression of oxidative stress regulatory genes, reducingstress induced by lipids, glucose, inflammatory cytokines orenvironmental ROS, e.g., via STAT1- and STAT3-mediated upregulation ofanti-oxidant genes and suppression of oxidative stress-inducing genes,reducing ER stress, promoting secretion of high quality efficaciousinsulin, restoring glucose homeostasis or enhancing peripheral insulinsensitivity.

In some embodiments, the IL-22 or IL-10 polypeptide is a biologicallyactive fragment of a full-length IL-22 or IL-10 polypeptide, (i.e., onethat includes less amino acids than the full-length IL-22 or IL-10polypeptide as set forth for example in SEQ ID NO: 2, 4, 6 or 8), andexhibits at least one activity selected from: improving pancreatic ERfunction, improving insulin biosynthesis, increasing glucose tolerance,inhibiting expression of oxidative stress regulatory genes, reducingstress induced by lipids, inflammatory cytokines or environmental ROS,e.g., via STAT1- and STAT3-mediated upregulation of anti-oxidant genesand suppression of oxidative stress-inducing genes, reducing ER stress,promoting secretion of high quality efficacious insulin, restoringglucose homeostasis or enhancing peripheral insulin sensitivity. Inspecific embodiments, the biologically active fragment comprises 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50 fewer residues at one or both ends (i.e., N-terminusand/or C-terminus) relative to the amino acid sequence of thefull-length polypeptide. In illustrative examples of this type, thebiologically active fragment comprises about 10 to about 145 amino acidresidues (and all integer amino acid residues in between).

2.2 Antagonists of OER Stress Inducers

The present inventors have found that IL-24, IL-23 and IL-33 are potentinducers of β-cell OER stress and that IL-18, MIP-2α, IL-17A, IFN-γ andIFN-β cause milder OER stress in a β-cell. Accordingly, it is proposedthat agents that inhibit or reduce the level or antagonize the functionof any one or more of these cytokines will reduce OER stress in aβ-cell. Illustrative examples of OER stress inhibitors of this typeinclude antigen-binding molecules that are immuno-interactive with anOER-stress inducing cytokine selected from the group consisting ofIL-24, IL-23, IL-33, IL-18, MIP-2α, IL-17A, IFN-γ and IFN-β, or solublereceptors that bind these cytokines.

In specific embodiments, the OER stress inhibitor is an antigen-bindingmolecule that is immuno-interactive with IL-24 (also known as melanomadifferentiation associated-7, or MDA-7). Antigen-binding molecules ofthis type are commercially available for example from Santa CruzBiotechnology (Santa Cruz, Calif.), LifeSpan Biosciences (Seattle,Wash.) and Proteintech Group (Chicago, Ill.).

In other embodiments, the OER stress inhibitor is a soluble receptorthat binds to IL-24, representative examples of which are disclosed inU.S. Pat. No. 7,855,269, which is hereby incorporated by referenceherein in its entirety. Suitably, the soluble receptor comprises anextracellular domain of IL-20RA and an extracellular domain of IL-20RBand in illustrative examples of this type, the extracellular domain ofIL-20RA comprises, consists or consists essentially of the amino acidsequence:VPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEFKAK [SEQ ID NO: 196], and wherein the extracellulardomain of IL-20RB comprises, consists or consists essentially of theamino acid sequence:DEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEAIP [SEQID NO: 197]. In some embodiments, the soluble receptor comprises (1) anextracellular domain of IL-20RA fused to the heavy chain of IgGy1, whichcomprises, consists or consists essentially of the sequence:VPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [SEQ ID NO: 198], and (2) an extracellular domain of IL-20RBfused to the human K light chain, which comprises, consists or consistsessentially of the sequence:DEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 199]. In theseembodiments, polypeptides (1) and (2) come together and a disulfide bondis formed between the heavy and the light chains to form a heterodimer.

In still other embodiments, the OER stress inhibitor is anantigen-binding molecule that is immuno-interactive with IL-23. Numerousantigen-binding molecules of this type are known, illustrative examplesof which include the anti-human IL-23 antigen-binding proteins disclosedin U.S. Pat. Appl. No. 2013/0004501, which is incorporated by referenceherein in its entirety. Exemplary antigen-binding proteins comprise aheavy chain variable region comprising complementarity determiningregions CDRH1, CDRH2, CDRH3 and a light chain variable region comprisingcomplementarity determining regions CDRL1, CDRL2, CDRL3 as defined inTable 3 of U.S. 2013/0004501. In one embodiment, the antigen-bindingprotein comprises a heavy chain variable domain of SEQ ID NO: 200(QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAFDIWGQGTMVTVSS), and alight chain variable domain of SEQ ID NO: 201(QSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFS GSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLTVL). A non-limiting example ofthis type is a single chain antibody that comprises the sequence:QSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLTVLGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAFDIWGQGTMVTVSS [SEQ ID NO;202].

In other embodiments, the OER stress inhibitor is an antigen-bindingmolecule that is immuno-interactive with IL-23R. Several antigen-bindingmolecules of this type are known, illustrative examples of which includethe anti-human IL-23R binding compounds disclosed in U.S. Pat. No.8,309,085, which is incorporated by reference herein in its entirety.Representative compounds of this type comprise: a) an antibody lightchain variable domain, or antigen binding fragment thereof, comprisingCDRL1, CDRL2 and CDRL3, wherein: CDRL1 comprises the sequence of SEQ IDNO: 203 (LASEDIYNNLA); CDRL2 comprises the sequence of SEQ ID NO: 204(HASSLQD); and CDRL3 comprises the sequence of SEQ ID NO: 205(LQDSEYPPT); and b) an antibody heavy chain variable domain, or antigenbinding fragment thereof, comprising CDRH1, CDRH2 and CDRH3, wherein:CDRH1 comprises the sequence of SEQ ID NO: 206 (GFDFNSYGMS); CDRH2comprises the sequence of SEQ ID NO: 207 (DINSKSYNYATYYADSVKD); andCDRH3 comprises the sequence of SEQ ID NO: 208 (HHSDYFEY). Inillustrative examples of this type, the anti-human IL-23R bindingcompound comprises a) an antibody light chain variable domain comprisingthe sequence of SEQ ID NO: 209(DIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKR); and b) an antibody heavychain variable domain comprising the sequence of SEQ ID NO: 210(QVQLVESGGGVVQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADINSKSYNYATYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTVSS). A non-limitingexample of this type is a single chain antibody that comprises thesequence:DIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADINSKSYNYATYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTVSS [SEQ ID NO: 211].

C3. Agents that Target the OER Stress Inhibitor to β-Cells

The present inventors propose that targeting β-cell OER stressinhibitors directly to β-cells will be useful for treating diseasesassociated with aberrant β-cell OER stress, suitably with reducedoff-target effects. In some embodiments, a β-cell OER stress inhibitoris associated with an agent that binds to a β-cell protein (e.g., aβ-cell surface protein) that is more highly expressed on β-cells thanone or more other cell types in the subject (e.g., epidermal cells,intestinal epithelial cells or hepatocytes). Non-limiting examples ofβ-cell proteins of this type and agents that bind to such proteins aredescribed below.

3.1 Glucagon-Like Peptide 1 Receptor

The glucagon-like peptide 1 receptor (GLP-1R) is encoded by the GLP1Rgene, which is highly expressed by β-cells, and expressed at very lowlevels in the skin and liver. Upon activation, GLP-1R stimulates theadenylyl cyclase pathway, resulting in increased insulin synthesis andrelease of insulin.

The natural agonist of GLP-1R is the peptide GLP-1, encoded by theGlucagon (GCG) gene. Preproglucagon is cleaved to produce two activeGLP-1 peptides GLP-17-36, and GLP-16-36.

Several GLP-1 analogs are currently in therapeutic use for the treatmentof type 2 diabetes, for example, exenatide and liraglutide. Thesemolecules promote glucose-dependent insulin secretion, due to favorableeffects on β-cell function, including suppression of ER stress.Accordingly, the use of GLP-1 and its analogs to target an OER stressinhibitor to a β-cell has the ability to provide dual benefits viaGLP-1R activation.

Endogenous GLP-1 has a short half-life due to the actions of thedipeptidyl peptidase-IV (DPP-4) protease. Long-acting relativelyprotease resistant forms of GLP-1 are known, and can suitably be fusedto or otherwise conjugated directly or indirectly to an OER stressinhibitor.

Non-limiting examples of GLP-1R agonists that are suitable for use as8-cell targeting agents include peptides or polypeptides comprising,consisting or consisting essentially of an amino acid sequence selectedfrom:

-   -   (a) an amino acid sequence selected from:        HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRR [SEQ ID NO: 10], which        corresponds to the amino acid sequence of native GLP-1,    -   HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG [SEQ ID NO: 12], which        corresponds to the amino acid sequence of GLP-11-37,    -   HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR [SEQ ID NO: 14], which        corresponds to the amino acid sequence of GLP-17-36,    -   HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG [SEQ ID NO: 16], which        corresponds to the amino acid sequence of GLP-17-37,    -   MKSIYFVAGLFVMLVQGSWQRSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTS        DYSKYLDSRRAQDFVQWLMNTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLV        KGRGRRDFPEEVAIVEELGRRHADGSFSDEMNTILDNLAARDFINWLIQTKITDRK [SEQ ID        NO: 18], which corresponds to the amino acid sequence of        glucagon preproprotein,    -   RSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSKYLDSRRAQDFVQWLM        NTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAIVEELGR        RHADGSFSDEMNTILDNLAARDFINWLIQTKITDRK [SEQ ID NO: 20], which        corresponds to the amino acid sequence of glucagon proprotein,    -   HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS [SEQ ID NO: 22], which        corresponds to the amino acid sequence of Exendin-4, or    -   MVATKTFALLLLSLFLAVGLGEKKEGH FSALPSLPVGSHAKVSSPQPRGPRYAEGTFISDYSI        AMDKIHQQDFVNWLLAQKGKKNDWKHNITQREARALELASQANRKEEEAVEPQSSPAKNPSDE        DLLRDLLIQELLACLLDQTNLCRLRSR [SEQ ID NO: 24], which corresponds        to the amino acid sequence of gastric inhibitory polypeptide        preproprotein,    -   (b) an amino acid sequence that shares at least 70% (and at        least 71% to at least 99% and all integer percentages in        between) sequence similarity or sequence identity with the        sequence set forth in any one of SEQ ID NO: 10, 12, 14, 16, 18,        20, 22 or 24; or    -   (c) an amino acid sequence that is encoded by a nucleotide        sequence selected from:        cacgatgaatttgagagacatgctgaagggacctttaccagtgatgtaagttcttatttggaaggccaagctgccaaggaa        ttcattgcttggctggtgaaaggccgaggaaggcga [SEQ ID NO: 9], which        corresponds to the coding sequence for the amino acid sequence        set forth in SEQ ID NO: 10, or    -   cacgatgaatttgagagacatgctgaagggacctttaccagtgatgtaagttcttatttggaaggccaagctgccaa        ggaattcattgcttggctggtgaaaggccgagga [SEQ ID NO: 11], which        corresponds to the coding sequence for the amino acid sequence        set forth in SEQ ID NO: 12,    -   catgctgaagggacctttaccagtgatgtaagttcttatttggaaggccaagctgccaaggaattcattgcttggctg        gtgaaaggccga [SEQ ID NO: 13], which corresponds to the coding        sequence for the amino acid sequence set forth in SEQ ID NO: 14,    -   catgctgaagggacctttaccagtgatgtaagttcttatttggaaggccaagctgccaaggaattcattgcttggctg        gtgaaaggccgagga [SEQ ID NO: 15], which corresponds to the coding        sequence for the amino acid sequence set forth in SEQ ID NO: 16,    -   atgaaaagcatttactttgtggctggattatttgtaatgctggtacaaggcagctggcaacgttcccttcaagacaca        gaggagaaatccagatcattctcagcttcccaggcagacccactcagtgatcctgatcagatgaacgaggacaagcgcc        attcacagggcacattcaccagtgactacagcaagtatctggactccaggcgtgcccaagattttgtgcagtggttgatga        ataccaagaggaacaggaataacattgccaaacgtcacgatgaatttgagagacatgctgaagggacctttaccagtga        tgtaagttcttatttggaaggccaagctgccaaggaattcattgcttggctggtgaaaggccgaggaaggcgagatttccc        agaagaggtcgccattgttgaagaacttggccgcagacatgctgatggttctttctctgatgagatgaacaccattcttgat        aatcttgccgccagggactttataaactggttgattcagaccaaaatcactgacaggaaa[SEQ        ID NO: 17], which corresponds to the coding sequence for the        amino acid sequence set forth in SEQ ID NO: 18,    -   cgttcccttcaagacacagaggagaaatccagatcattctcagcttcccaggcagacccactcagtgatcctgatca        gatgaacgaggacaagcgccattcacagggcacattcaccagtgactacagcaagtatctggactccaggcgtgcccaa        gattttgtgcagtggttgatgaataccaagaggaacaggaataacattgccaaacgtcacgatgaatttgagagacatgc        tgaagggacctttaccagtgatgtaagttcttatttggaaggccaagctgccaaggaattcattgcttggctggtgaaaggc        cgaggaaggcgagatttcccagaagaggtcgccattgttgaagaacttggccgcagacatgctgatggttctttctctgat        gagatgaacaccattcttgataatcttgccgccagggactttataaactggttgattcagaccaaaatcactgacaggaaa        [SEQ ID NO: 19], which corresponds to the coding sequence for        the amino acid sequence set forth in SEQ ID NO: 20,    -   catggcgaaggcacctttaccagcgatctgagcaaacagatggaagaagaagcggtgcgcctgtttattgaatggc        tgaaaaacggcggcccgagcagcggcgcgccgccgccgagc [SEQ ID NO: 21], which        corresponds to the coding sequence for the amino acid sequence        set forth in SEQ ID NO: 22, or    -   catggcgaaggcacctttaccagcgatctgagcaaacagatggaagaagaagcggtgcgcctgtttattgaatggc        tgaaaaacggcggcccgagcagcggcgcgccgccgccgagc [SEQ ID NO: 23], which        corresponds to the coding sequence for the amino acid sequence        set forth in SEQ ID NO: 24;    -   (d) an amino acid sequence that is encoded by a nucleotide        sequence that shares at least 70% (and at least 71% to at least        99% and all integer percentages in between) sequence identity        with the sequence set forth in SEQ ID NO: 9, 11, 13, 15, 17, 19,        21 or 23, or a complement thereof; or    -   (e) an amino acid sequence that is encoded by a nucleotide        sequence that hybridizes under low, medium or high stringency        conditions to the sequence set forth in any one of SEQ ID NO: 9,        11, 13, 15, 17, 19, 21 or 23, or a complement thereof,    -   wherein the amino acid sequence of (a), (b), (c), (d) or (e) is        an agonist of GLP-1R.

In specific embodiments, the GLP-1 agonist is selected from:

-   -   (1) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2 [SEQ ID NO: 25],        which is an amidated Exendin-4 analog (Exenatide, BYETTA);    -   (2) H(2-methyl)-AEGTFTSDVSSYLEGQAAKEFIAWLVK(2-methyl)AR-CONH2        [SEQ ID NO: 26] (Taspoglutide);    -   (3) HAEGTFTSDVSSYLEGQAA-K(E-palmitic acid)-DEFIAWLVRGRG [SEQ ID        NO: 27] (Liraglutide, VICTOZA); or    -   (4) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2 [SEQ ID NO:        28] (Lixisenatide, LYXUMIA).    -   (5) HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRHGEGTFTSDVSSYLEGQAAKEFIAWLV        KGRMKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFED        HVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL        QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECC        QAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKL        VTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMP        ADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAAD        PHECYAKVFDEFKPLVEEPQN        LIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRN LG        KVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET        YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKAD        DKETCFAEEGKKLVAASQAALGL [SEQ ID NO: 29], which is a GLP-1 dimer        fused to albumin (Albiglutide, EPERZAN TANZEUM).    -   (6) a GLP-1 analog selected from        HX8EGTFTSDVSSYLEGQAAKEFIAWLVKGGG [SEQ ID NO: 30], wherein Xs is        selected from G or V; HX₈EGTFTSDVSSYLEGQAAKEFIAWLKNGGG [SEQ ID        NO: 31], wherein Xs is selected from G or V;        HX₈EGTFTSDVSSYLEGQAAKEFIAWLVKGGP [SEQ ID NO: 32], wherein Xs is        selected from G or V; HX₈EGTFTSDVSSYLEGQAAKEFIAWLKNGGP [SEQ ID        NO: 33], wherein Xs is selected from G or V;        HX₈EGTFTSDVSSYLEGQAAKEFIAWLVKGG [SEQ ID NO: 34], wherein Xs is        selected from G or V; or HX₈EGTFTSDVSSYLEGQAAKEFIAWLKNGG [SEQ ID        NO: 35], wherein Xs is selected from G or V, wherein the GLP-1        analog is fused to the Fc portion of an immunoglobulin        comprising the sequence        AESKYGPPCPPCPAPX₁₆X₁₇X₁₅GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV        DGVEVHNAKTKPREEQFX₈₀STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ        PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY        SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGX₂₃₀ [SEQ ID NO: 36],        wherein X₁₆ is selected from P or E, X₁₇ is selected from F, V        or A, Xis is selected from L, E or A, and X₂₃₀ is K or is        absent.

Alternatively, the GLP-1R agonist may be selected from small molecules,illustrative examples of which include hydroxylflavonols such as 4′hydroxylflavonol, 3′,4′ hydroxylflavonol, and quercetin.

In other embodiments, the GLP-1R binding agent is an agonisticantigen-binding molecule that is immuno-interactive with GLP-1R.Representative antigen-binding molecules of this type include monoclonalantibody 5A10, as described, for example, in US 2006/0275288, which ishereby incorporated by reference herein in its entirety, as well asantigen-binding fragments of monoclonal antibody 5A10 and humanized orchimeric antibodies and antigen-binding fragments thereof. Monoclonal5A10 antibody comprises:

the light chain variable region amino acid sequence:

[SEQ ID NO: 37] QIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGG GTRLEIKR; andthe heavy chain variable region amino acid sequence:

[SEQ ID NO: 38] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDYWGQGTSVTVSS. 

Also contemplated are antibodies and antigen-binding fragments thatcomprise the light chain and heavy chain complementarity determiningregion (CDR) sequences of the 5A10 monoclonal antibody, wherein thelight chain CDR sequences comprise CDR1 of SEQ ID NO: 39 (SASSRVTYMH);CDR2 of SEQ ID NO: 40 (DTSKLAS); and CDR3 of SEQ ID NO: 41 (QQWGNNPQYT),and the heavy chain CDR sequences comprise CDR1 of SEQ ID NO: 42(GFSLSTSGTGVG); CDR2 of SEQ ID NO: 43 (HIWWDDVKRYNPALKS) and CDR3 of SEQID NO: 44 (ILDGTGPMDY). In specific embodiments, these antibodies orantigen-binding fragments comprise human antibody framework (FR) andconstant region sequences with one or more framework region amino acidresidues substituted from the corresponding framework region sequencesof the parent 5A10 antibody.

In illustrative examples of this type, the antigen-binding molecule is asingle chain Fv (scFv) antibody comprising, consisting or consistingessentially of the amino acid sequence:

[SEQ ID NO: 45] QIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDYWGQGTSVTVSS.

3.2 Sulfonylurea Receptor

The sulfonylurea receptor is an inward-rectifier potassium ion channelthat is highly expressed by 3-cells. The ATP-sensitive K⁺ channelreceptor is composed from two major subunits: SUR1 (encoded by the geneABCC8) and Kir6.2 (encoded by the gene KCNJ11).

Kir6.2 is also expressed in cardiac and skeletal muscle. In theintestine expression of Kir6.2 is restricted to relatively rareenteroendocrine cells, L-cells and K-cells. The receptor is the targetfor a broadly used class of type 2 diabetes therapeutics, thesulfonylureas, for example GLIPIZIDE, GLICLAZIDE and GLIBENCLAMIDE,which drive insulin secretion in a glucose-independent manner.

Notably, it is only essential that the sulfonylurea bind thesulfonylurea receptor as it is used primarily to target the OER stressinhibitor to the 3-cell. Thus, it is not necessary for the sulfonylureato exert its biological activity. In fact, in accordance with thepresent invention, the OER stress inhibitor IL-22 is administered to asubject at a concentration of between 20-100 μg/kg (1.25-6 nmoles/kg).On the other hand, the sulfonylureas are administered at a concentrationof between 10-200 mg/kg (˜30-600 μmoles/kg). Therefore, the sulfonylureain the conjugate will have virtually no activity due to the lowconcentration.

Non-limiting examples of sulfonylurea receptor agonists that aresuitable for use with the present invention include:

α-endosulfine:

[SEQ ID NO: 46] MSQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTP QDLPQRKSSLVTSKLAGGQV;

3.3 Free fatty acid receptor 1 (FFA1)

Free fatty acid receptor 1 (FFA1), also known as GPR40, is a class AG-protein coupled receptor that in humans is encoded by the FFAR1 gene.It is strongly expressed in the cells of the pancreas and to a lesserextent in the brain. This membrane protein binds free fatty acids,acting as a nutrient sensor for regulating energy homeostasis. Agonistsof this receptor are useful for reducing HbA1c in type 2 diabetespatients. Accordingly, the present invention also contemplatesconjugating GPR40 agonists to an OER stress inhibitor. Non-limitingexamples of such agonists include:

3.4G Protein-Coupled Receptor 119 (GPR119)

GPR119 is expressed predominantly in the pancreas and gastrointestinaltract in rodents and humans, as well as in the brain in rodents.Activation of the receptor has been shown to cause insulin release inβ-cells, a reduction in food intake and body weight gain in rats. GPR119has also been shown to regulate incretin and insulin hormone secretion.As a result, drugs acting on the receptor have been developed fortreatment of obesity and diabetes. Thus, the present invention alsocontemplates conjugating GPR119 agonists to an OER stress inhibitor.Illustrative agonists of this type include:

Alternative GPR119 agonists are described in X.-Y. Ye et al. (2014,Bioorg. Med. Chem. Lett. 24 (2014) 2539-2545), which is herebyincorporated by reference herein in its entirety.

3.5 Unspecified β-Cell Surface Antigen

In some embodiments, the β-cell targeting agent is an antigen-bindingmolecule that is immuno-interactive with an unspecified/uncharacterizedantigen on the surface of pancreatic β-cells, as disclosed for examplein WO 2010/096930, the contents of which are hereby incorporated hereinin their entirety. These antigen-binding molecules bind selectively topancreatic β-cells and comprise a heavy chain variable sequence selectedfrom:

[SEQ ID NO: 47] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTS YRFDYWGQGTLVT;[SEQ ID NO: 48] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSRIKIFGSKTKFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHS THFDYWGQGTLVT;[SEQ ID NO: 49] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIHPKGYPTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKST TPFDYWGQGTLVT;[SEQ ID NO: 50] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSRIQFFGSHTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHS THFDYWGQGTLVT;[SEQ ID NO: 51] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSTGDSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAA DSFDYWGQGTLVT,

and a light chain variable region selected from:

[SEQ ID NO: 52] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQ GTKVEIKR;[SEQ ID NO: 53] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLQSTPRTFGQ GTKVEIKR;[SEQ ID NO: 54] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQMGRDPRTFGQ GTKVEIKR;[SEQ ID NO: 55] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNRRIPRTFGQ GTKVEIKR;[SEQ ID NO: 56] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTNGAPTTFGQ GTKVEIKR.

In specific embodiments, antigen-binding molecules of this type aresingle chain antibodies (e.g., scFv) comprising an amino acid sequenceselected from:

[SEQ ID NO: 57] AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; [SEQ ID NO: 58]AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSRIKIFGSKTKFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHSTHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLQSTPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; [SEQ ID NO: 59]AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIHPKGYPTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTTPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQMGRDPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; [SEQ ID NO: 60]AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSRIQFFGSHTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHSTHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNRRIPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; or [SEQ ID NO: 61]AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSTGDSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAADSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTNGAPTTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN.

Also contemplated are antibodies and antigen-binding fragments thatcomprise the light chain and heavy chain complementarity determiningregion (CDR) sequences of the heavy and light chain variable regionslisted above, wherein the heavy chain CDR sequences comprise CDR1 of SEQID NO: 62 (SYAMS); a CDR2 selected from SEQ ID NO: 63(SITAEGTHTWYADSVKG), SEQ ID NO: 64 (RIKIFGSKTKFADSVKG), SEQ ID NO: 65(SIHPKGYPTRYADSVKG), SEQ ID NO: 66 (RIQFFGSHTYFADSVKG) or SEQ ID NO: 67(SISSTGDSTSYADSVKG); and a CDR3 selected from SEQ ID NO: 68 (TSYRFDY),SEQ ID NO: 69 (HSTHFDY), SEQ ID NO: 70 (STTPFDY) or SEQ ID NO: 71(AADSFDY), and the light chain CDR sequences comprise CDR1 of SEQ ID NO:72 (RASQSISSYLN); a CDR2 selected from SEQ ID NO: 73 (KASRLQS), SEQ IDNO: 74 (RASSLQS), SEQ ID NO: 75 (AASSLQS), SEQ ID NO: 76 (RASILQS), orSEQ ID NO: 77 (GASSLQS), and a CDR3 selected from SEQ ID NO: 78(QQKWDPPRT), SEQ ID NO: 79 (QQLQSTPRT), SEQ ID NO: 80 (QQMGRDPRT), SEQID NO: 81 (QQNRRIPRT) or SEQ ID NO: 82 (QQTNGAPTT).

In illustrative examples of this type, the antibody or antigen-bindingfragments that comprise:

heavy chain CDR sequences comprising a CDR1 of SEQ ID NO: 62 (SYAMS); aCDR2 of SEQ ID NO: 63 (SITAEGTHTWYADSVKG) and a CDR3 of SEQ ID NO: 68(TSYRFDY), and light chain CDR sequences comprising a CDR1 of SEQ ID NO:72 (RASQSISSYLN); a CDR2 of SEQ ID NO: 73 (KASRLQS) and a CDR3 of SEQ IDNO: 78 (QQKWDPPRT);

heavy chain CDR sequences comprising a CDR1 of SEQ ID NO: 62 (SYAMS); aCDR2 of SEQ ID NO: 65 (SIHPKGYPTRYADSVKG) and a CDR3 of SEQ ID NO: 69(HSTHFDY), and light chain CDR sequences comprising a CDR1 of SEQ ID NO:72 (RASQSISSYLN); a CDR2 of SEQ ID NO: 74 (RASSLQS) and a CDR3 of SEQ IDNO: 79 (QQLQSTPRT);

heavy chain CDR sequences comprising a CDR1 of SEQ ID NO: 62 (SYAMS); aCDR2 of SEQ ID NO: 64 (RIKIFGSKTKFADSVKG) and a CDR3 of SEQ ID NO: 70(STTPFDY), and light chain CDR sequences comprising a CDR1 of SEQ ID NO:72 (RASQSISSYLN); a CDR2 of SEQ ID NO: 75 (AASSLQS) and a CDR3 of SEQ IDNO: 80 (QQMGRDPRT);

heavy chain CDR sequences comprising a CDR1 of SEQ ID NO: 62 (SYAMS); aCDR2 of SEQ ID NO: 66 (RIQFFGSHTYFADSVKG) and a CDR3 of SEQ ID NO: 69(HSTHFDY), and light chain CDR sequences comprising a CDR1 of SEQ ID NO:72 (RASQSISSYLN); a CDR2 of SEQ ID NO: 76 (RASILQS) and a CDR3 of SEQ IDNO: 81 (QQNRRIPRT); or

heavy chain CDR sequences comprising a CDR1 of SEQ ID NO: 62 (SYAMS); aCDR2 of SEQ ID NO: 67 (SISSTGDSTSYADSVKG) and a CDR3 of SEQ ID NO: 71(AADSFDY), and light chain CDR sequences comprising a CDR1 of SEQ ID NO:72 (RASQSISSYLN); a CDR2 of SEQ ID NO: 77 (GASSLQS) and a CDR3 of SEQ IDNO: 82 (QQTNGAPTT).

In specific embodiments, the above antibodies or antigen-bindingfragments comprise human antibody framework (FR) and constant regionsequences with one or more framework region amino acid residuessubstituted from the corresponding framework region sequences of theparent scFv antibodies SCA B1, SCA B2, SCA B3, SCA B4 or SCA B5, astaught in WO 2010/096930.

Alternative β-cell specific antibodies and methods for their preparationare described, for example, by Sung et al. (Mol Cancer Ther. 2009August; 8(8):2276-85), Feng et al. (MAbs. 2010 September-October;2(5):565-70), Chen et al. (Cell Research (2009) 19:984-995).

4. Conjugation/Fusion

The present invention contemplates any suitable method for conjugatingor fusing β-cell binding agents to OER stress inhibitors, non-limitingexamples of which include:

-   -   (i) Use of a carbodiimide or other suitable coupling agent to        form an amide linkage between a free amino group of a β-cell        binding agent and the free carboxyl group of an OER stress        inhibitor protein or a linker peptide fused or otherwise        attached to the OER stress inhibitor protein;    -   (ii) Use of a carbodiimide or other suitable coupling agent to        form an amide linkage between a free amino group of an OER        stress inhibitor protein and the free carboxyl group of a β-cell        binding agent or a linker peptide fused or otherwise attached to        the β-cell binding agent;    -   (iii) Use of MBS (m-maleimidonemzoic acid N-hydroxysuccinimide        ester) to link via the thiol group of a cysteine added to the        C-terminus of an OER stress inhibitor protein or to the        C-terminus of a linker peptide attached or otherwise fused to an        OER stress inhibitor protein, to the free amino group at the        N-terminus of the a β-cell binding protein;    -   (iv) Use of MBS (m-maleimidonemzoic acid N-hydroxysuccinimide        ester) to link via the thiol group of a cysteine added to the        C-terminus of a β-cell binding agent (suitably a β-cell binding        protein) or to the C-terminus of a linker peptide attached or        otherwise fused to a β-cell binding agent (suitably a β-cell        binding protein), to the free amino group at the N-terminus of        the an OER stress inhibitor protein;    -   (v) Use of glutaraldehyde to conjugate a free amino group on an        OER-stress inhibitor protein or on a linker protein fused or        otherwise attached to an OER-stress inhibitor protein and a free        amino group on a β-cell binding agent (suitably a β-cell binding        protein) or on a linker protein fused or otherwise attached to a        β-cell binding agent (suitably a β-cell binding protein);    -   (vi) Use of SO₂Cl or triazoles to couple an amine of an        OER-stress inhibitor protein to an aldehyde-containing moiety of        a β-cell binding agent (suitably a β-cell binding protein); or    -   (vii) Use of SO₂Cl or triazoles to couple an amine of a β-cell        binding agent (suitably a β-cell binding protein) to an        aldehyde-containing moiety of an OER-stress inhibitor protein.

In illustrative examples of conjugating sulfonylurea compounds withamino or carboxy groups to an OER stress inhibitor protein, thesecompounds can be conjugated to the C- or N-terminus of the protein,either with or without linker peptides extending from those termini, byrecombinant fusion or by peptide-peptide covalent conjugation. Forinstance, such conjugates may be made through: (a) use of a carbodiimideto form an amide linkage between a free amino group on theamino-sulfonylureas and the free carboxyl group on the C-terminus of OERstress inhibitor or the spacer peptide; or (b) use of a carbodiimide toform an amide linkage between a free carboxy group on thecarboxy-sulfonylureas and the free amino group on the N-terminus of theOER stress inhibitor or the spacer peptide. Linker peptides could beprepared with several spaced reactive residues via which to conjugatemultiple sulfonylurea compounds.

In specific embodiments, conjugates/fusions of a β-cell binding proteinand an OER stress inhibitor protein, with or without interveninglinkers, can be made by peptide synthesis or by recombinant expressionusing any suitable prokaryotic or eukaryotic expression system(including but not limited to bacterial, yeast, insect and mammaliancells), as known in the art.

5. Chimeric Constructs

One aspect of the present invention relates to chimeric constructs thatcomprise an OER stress inhibitor polypeptide that is fused to orotherwise conjugated, either directly or via a linker, to aproteinaceous β-cell targeting agent. In specific embodiments, the OERstress inhibitor is an IL-22 polypeptide and the targeting agent is aproteinaceous GLP-1R agonist. Illustrative constructs may comprise smallGLP-1 peptides and peptide analogs, representative examples of whichinclude:

(a) [SEQ ID NO: 83] MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRR[GGGGSGGGGSGGGGSGGGGS]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (b) [SEQ ID NO: 84]MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRR [GGGSGGGS]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGE LDLLFMSLRNACI; (C)[SEQ ID NO: 85] MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRR[GGGG]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNA CI; (d) [SEQ ID NO: 86]MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRR[GGGGG]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNA CI; (e) [SEQ ID NO: 87]MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRR [GGGKGGGG]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGE LDLLFMSLRNACI; (f)[SEQ ID NO: 88] MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRR [GGGNGSGG]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGE LDLLFMSLRNACI; (g)[SEQ ID NO: 89] MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRR [GGGCGGGG]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGE LDLLFMSLRNACI; (h)[SEQ ID NO: 90] MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRR[GPNGG]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNA CI; and (i)[SEQ ID NO: 91] MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI, wherein:HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRR = native  GLP-1;Underlined text is flexible linker;Text in italics is IL-22 mature polypeptide;M is an artificial methionine for recombinant  expression;n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8,  9 or 10; andX is an optional tag (e.g., FLAGs, His₆) for purification, or (j)[SEQ ID NO: 92] MX APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI[GGGGSGGGGSGGGGSGGGGS]_(n) HDEFERHAEGTFTSDVSSYLE GQAAKEFIAWLVKGRGRR; (k)[SEQ ID NO: 93] MX APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GGGSGGGS]_(n)HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLV  KGRGRR; (l) [SEQ ID NO: 94] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GGGG]_(n)HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG RR; (m) [SEQ ID NO: 95] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GGGGG]_(n)HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGR GRR; (n) [SEQ ID NO: 96] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GGGKGGGG]_(n)HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLV  KGRGRR; (o) [SEQ ID NO: 97] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GGGNGSGG]_(n)HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLV KGRGRR; (p) [SEQ ID NO: 98] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GGGCGGGG]_(n)HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLV KGRGRR; (q) [SEQ ID NO: 99] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GPNGG]_(n)HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGR GRR; and (r) [SEQ ID NO: 100] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACIHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRR, wherein:HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRR = native GLP-1;Underlined text is flexible linker;Text in italics is IL-22 mature polypeptide;M is an artificial methionine for recombinant expression;n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; andX is an optional tag (e.g., FLAGs, His₆) for purification, or (s)[SEQ ID NO: 101] MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG[GGGGSGGGGSGGGGSGGGGS]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (t) [SEQ ID NO: 102]MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG [GGGGSGGGS]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIG ELDLLFMSLRNACI; (u)[SEQ ID NO: 103] MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG[GGGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (v) [SEQ ID NO: 104]MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG[GGGGGG]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNA CI; (w)[SEQ ID NO: 105] MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG [GGGGKGGGG]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIG ELDLLFMSLRNACI; (x)[SEQ ID NO: 106] MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG [GGGNGSGG]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEWPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGEL DLLFMSLRNACI; (y)[SEQ ID NO: 107] MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG [GGGGCGGGG]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIG ELDLLFMSLRNACI; (z)[SEQ ID NO: 108] MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG[GPNGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNAC I; and (aa)[SEQ ID NO: 109] MXHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNAC, wherein:HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG = GLP-1₁₋₃₇;Underlined text is flexible linker;Text in italics is IL-22 mature polypeptide;M is an artificial methionine for recombinant expression;n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; andX is an optional tag (e.g., FLAGs, His₆) for purification, or (ab)[SEQ ID NO: 110] MX APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI[GGGGSGGGGSGGGGSGGGGS]_(n)HDEFERHAEGTFTSDVSSYLE GQAAKEFIAWLVKGRG; (ac)[SEQ ID NO: 111] MX APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR  NACI [GGGSGGGS]_(n)HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLV KGRG; (ad) [SEQ ID NO: 112] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GGGG]_(n)HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG; (ae) [SEQ ID NO: 113] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GGGGG]_(n)HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGR G; (af) [SEQ ID NO: 114] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GGGKGGGG]_(n)HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLV  KGRG; (ag) [SEQ ID NO: 115] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GGGNGSGG]_(n)HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLV KGRG; (ah) [SEQ ID NO: 116] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GGGCGGGG]_(n)HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLV  KGRG; (ai) [SEQ ID NO: 117] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GPNGG]_(n)HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGR G; and (aj) [SEQ ID NO: 118] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACIHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG, wherein:HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG = GLP-1₁₋₃₇;Underlined text is flexible linker;Text in italics is IL-22 mature polypeptide;M is an artificial methionine for recombinant expression;n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; andX is an optional tag (e.g., FLAGs, His₆) for purification, or (ak)[SEQ ID NO: 119] MXHAEGTFTSDVSSYLEGQAAKEFIAWLVKGR[GGGGSGGGGSGGGGSGGGGS]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (al) [SEQ ID NO: 120]MXHAEGTFTSDVSSYLEGQAAKEFIAWLVKGR[GGGSGGGS]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (am) [SEQ ID NO: 121]MXHAEGTFTSDVSSYLEGQAAKEFIAWLVKGR[GGGGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (an) [SEQ ID NO: 122]MXHAEGTFTSDVSSYLEGQAAKEFIAWLVKGR[GGGGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (ao) [SEQ ID NO: 123]MXHAEGTFTSDVSSYLEGQAAKEFIAWLVKGR[GGGGKGGGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (ap) [SEQ ID NO: 124]MXHAEGTFTSDVSSYLEGQAAKEFIAWLVKGR[GGGNGSGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (aq) [SEQ ID NO: 125]MXHAEGTFTSDVSSYLEGQAAKEFIAWLVKGR[GGGCGGGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (ar) [SEQ ID NO: 126]MXHAEGTFTSDVSSYLEGQAAKEFIAWLVKGR[GPNGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; and (as) [SEQ ID NO: 127]MXHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI, wherein:HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR = GLP-1₇₋₃₆;Underlined text is flexible linker;Text in italics is IL-22 mature polypeptide;M is an artificial methionine for recombinant expression;n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; andX is an optional tag (e.g., FLAGs, His₆) for purification, or (at)[SEQ ID NO: 128] MXHAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSL  RNACI[GGGGSGGGGSGGGGSGGGGS]_(n) HAEGTFTSDVSSYLEGQAAK EFIAWLVKGR; (au)[SEQ ID NO: 129] MX APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GGGSGGGS]_(n)HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR; (av) [SEQ ID NO: 130] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GGGG]_(n)HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR; (aw) [SEQ ID NO: 131] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GGGGG]_(n)HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR; (ax) [SEQ ID NO: 132] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GGGKGGGG]_(n)HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR; (ay) [SEQ ID NO: 133] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GGGNGSGG]_(n)HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR; (az) [SEQ ID NO: 134] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GGGCGGGG]_(n)HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR; (aaa) [SEQ ID NO: 135] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI [GPNGG]_(n)HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR;  and (aab) [SEQ ID NO: 136] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACIHAEGTFTSDVSSYLEGQAAKEFIAWLVKGR, wherein:HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR = GLP-1₇₋₃₆;Underlined text is flexible linker;Text in italics is IL-22 mature polypeptide;M is an artificial methionine for recombinant  expression;n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8,  9 or 10; andX is an optional tag (e.g., FLAGs, His₆) for  purification.

In other embodiments, the chimeric constructs comprise alternative GLP-1peptide analogs substituted for the peptide analogs defined above,illustrative examples of which include:

(1) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG [SEQ ID NO: 16], which correspondsto the amino acid sequence of GLP-17-37,

(2) MKSIYFVAGLFVMLVQGSWQRSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAIVEELGRRHADGSFSDEMNTILDNLAARDFINWLIQTKITDRK [SEQ ID NO:18], which corresponds to the amino acid sequence of glucagonpreproprotein,

(3) RSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAIVEELGRRHADGSFSDEMNTILDNLAARDFINWLIQTKITDRK [SEQ ID NO: 20], which correspondsto the amino acid sequence of glucagon proprotein,

(4) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS [SEQ ID NO: 22], whichcorresponds to the amino acid sequence of Exendin-4,

(5) MVATKTFALLLLSLFLAVGLGEKKEGHFSALPSLPVGSHAKVSSPQPRGPRYAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQREARALELASQANRKEEEAVEPQSSPAKNPSDEDLLRDLLIQELLACLLDQTNLCRLRSR [SEQ ID NO: 24], which corresponds to theamino acid sequence of gastric inhibitory polypeptide preproprotein, and

(6) a GLP-1 analog selected from HXsEGTFTSDVSSYLEGQAAKEFIAWLVKGGG [SEQID NO: 30], wherein Xs is selected from G or V;HX₈EGTFTSDVSSYLEGQAAKEFIAWLKNGGG [SEQ ID NO: 31], wherein Xs is selectedfrom G or V; HX₈EGTFTSDVSSYLEGQAAKEFIAWLVKGGP [SEQ ID NO: 32], whereinXs is selected from G or V; HX₈EGTFTSDVSSYLEGQAAKEFIAWLKNGGP [SEQ ID NO:33], wherein Xs is selected from G or V; HX₈EGTFTSDVSSYLEGQAAKEFIAWLVKGG[SEQ ID NO: 34], wherein Xs is selected from G or V; orHX₈EGTFTSDVSSYLEGQAAKEFIAWLKNGG [SEQ ID NO: 35], wherein Xs is selectedfrom G or V, wherein the GLP-1 analog is fused to the Fc portion of animmunoglobulin comprising the sequenceAESKYGPPCPPCPAPX₁₆X₁₇X₁₈GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFX₈₀STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGX230 [SEQ ID NO: 36], wherein X₁₆ isselected from P or E, X₁₇ is selected from F, V or A, Xis is selectedfrom L, E or A, and X₂₃₀ is K or is absent.

In other embodiments, the GLP-1R targeting agent of the chimericconstruct is an antigen-binding molecule and non-limiting constructs ofthis type include:

(i) [SEQ ID NO: 137] MXQIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDYWGQGTSVTVSS[GGGGSGGGGSGGGGSGGGGS]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (ii) [SEQ ID NO: 138]MXQIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDYWGQGTS VTVSS[GGGSGGGS]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAI GELDLLFMSLRNACI; (iii)[SEQ ID NO: 139] MXQIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDYWGQGTS VTVSS[GGGG]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELD LLFMSLRNACI; (iv)[SEQ ID NO: 140] MXQIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDYWGQGTS VTVSS[GGGGG]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGEL DLLFMSLRNACI; (v)[SEQ ID NO: 141] MXQIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDYWGQGTS VTVSS[GGGGKGGGG]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKA IGELDLLFMSLRNACI; (vi)[SEQ ID NO: 142] MXQIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDYWGQGTS VTVSS[GGGNGSGG]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAI GELDLLFMSLRNACI; (vii)[SEQ ID NO: 143] MXQIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDYWGQGTS VTVSS[GGGCGGGG]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAI GELDLLFMSLRNACI;(viii) [SEQ ID NO: 144]MXQIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDYWGQGTS VTVSS[GPNGG]_(n)APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGEL DLLFMSLRNACI; and (ix)[SEQ ID NO: 145] MXQIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDYWGQGTSVTVSSAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLR NACI, wherein:QIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDYWGQGTSVTVSS [SEQ ID NO: 45] = GLP-1R agonist scFv;Underlined text is flexible linker;Text in italics is IL-22 mature polypeptide;M is an artificial methionine for recombinant expression;n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8,  9 or 10; andX is an optional tag (e.g., FLAGs, His₆) for purification, or (x)[SEQ ID NO: 146] MXHAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNA CI[GGGGSGGGGSGGGGSGGGGS]_(n) QIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDYWGQGTSVTVSS; (xi) [SEQ ID NO: 147] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNAC I [GGGSGGGS]_(n)QIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTG PMDYWGQGTSVTVSS; (xii)[SEQ ID NO: 148] MX APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNAC I [GGGG]_(n)QIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDY WGQGTSVTVSS; (xiii)[SEQ ID NO: 149] MX APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNAC I [GGGGG]_(n)QIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMD YWGQGTSVTVSS; (xiv)[SEQ ID NO: 150] MX APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNAC I [GGGKGGGG]_(n)QIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTG PMDYWGQGTSVTVSS; (xv)[SEQ ID NO: 151] MX APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNAC I [GGGNGSGG]_(n)QIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTG PMDYWGQGTSVTVSS;(xvii) [SEQ ID NO: 152] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNAC I [GGGCGGGG]_(n)QIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTG PMDYWGQGTSVTVSS;(xviii) [SEQ ID NO: 153] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNAC I [GPNGG]_(n)QIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMD YWGQGTSVTVSS; and(xix) [SEQ ID NO: 154] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACIQIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDYWGQGTSV TVSS, wherein:QIVLTQSPAIMSASPGEKVTMTCSASSRVTYMHWYQQRSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGNNPQYTFGGGTRLEIKRGGGGSGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGTGVGWIRQPSGKGLEWLSHIWWDDVKRYNPALKSRLTISRDTSYSQVFLRIASVDTADTATYYCARILDGTGPMDYWGQGTSVTVSS [SEQ ID NO: 45] = GLP-1R agonist scFv,Underlined text is flexible linker;Text in italics is IL-22 mature polypeptide;M is an artificial methionine for recombinant expression;n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; andX is an optional tag (e.g., FLAGs, His₆) for purification.

In other embodiments, the OER stress inhibitor is an IL-22 polypeptideand the targeting agent is an antigen-binding molecule that isimmuno-interactive with a β-cell surface binding protein as taught forexample in WO 2010/096930. Non-limiting examples of constructs of thistype include:

(i) [SEQ ID NO: 155] MAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGGSGGGGSGGGGSGGGGS]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (ii) [SEQ ID NO: 156]MAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGSGGGS]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (iii) [SEQ ID NO: 157]MAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (iv) [SEQ ID NO: 158]MAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (v)  [SEQ ID NO: 159]MAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGGKGGGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (vi)  [SEQ ID NO: 160]MAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGNGSGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (vii) [SEQ ID NO: 161]MAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGCGGGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI; (viii) [SEQ ID NO: 162]MAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GPNGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI;  and (ix) [SEQ ID NO: 163]MAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLNAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGES GEIKAIGELDLLFMSLRNACI,wherein: AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN [SEQ ID NO: 57] = β-cell  specific scFv;Underlined text is flexible linker;Text in italics is IL-22 mature polypeptide;M is an artificial methionine for recombinant  expression; andn = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8,  9 or 10, or (x)[SEQ ID NO: 164] M APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI[GGGGSGGGGSGGGGSGGGGS]_(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; (xi) [SEQ ID NO: 165] MAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI [GGGSGGGS]_(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; (xii) [SEQ ID NO: 166] MAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI [GGGG]_(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; (xiii) [SEQ ID NO: 167] MAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI [GGGGG]_(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; (xiv) [SEQ ID NO: 168] MAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI [GGGKGGGG]_(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; (xv) [SEQ ID NO: 169] MAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI [GGGNGSGG]_(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; (xvii) [SEQ ID NO: 170] MAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI [GGGCGGGG]_(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; (xviii) [SEQ ID NO: 171] MAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI [GPNGG]_(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; and (xix) [SEQ ID NO: 172] MAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACIQIAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN, wherein:AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN [SEQ ID NO: 57] = β-cell specific scFv,Underlined text is flexible linker;Text in italics is IL-22 mature polypeptide;M is an artificial methionine for recombinant  expression; andn = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8,  9 or 10;

In other embodiments, the chimeric constructs comprise alternativeβ=cell specific scFv substituted for the scFv defined above,illustrative examples of which include:

[SEQ ID NO: 58] AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSRIKIFGSKTKFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHSTHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLQSTPRTFGQGTKVEIKRAA AHHHHHHGAAEQKLISEEDLN;[SEQ ID NO: 59] AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIHPKGYPTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTTPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQMGRDPRTFGQGTKVEIKRAA AHHHHHHGAAEQKLISEEDLN;[SEQ ID NO: 60] AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSRIQFFGSHTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHSTHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNRRIPRTFGQGTKVEIKRAAA HHHHHHGAAEQKLISEEDLN;and [SEQ ID NO: 61] AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSTGDSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAADSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTNGAPTTFGQGTKVEIKRAA AHHHHHHGAAEQKLISEEDLN.

In other embodiments, the OER stress inhibitor is an IL-22 polypeptideand the targeting agent is a proteinaceous SUR1 agonist such asα-endosulfine. Illustrative constructs of this type include:

(1) [SEQ ID NO: 173] MSQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV[GGGGSGGGGSGGGGSGGGGS]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI X; (2) [SEQ ID NO: 174]MSQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV[GGGSGGGS]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI X; (3) [SEQ ID NO: 175]MSQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV[GGGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI X; (4) [SEQ ID NO: 176]MSQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV[GGGGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI X; (5) [SEQ ID NO: 177]MSQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV[GGGKGGGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI X; (6) [SEQ ID NO: 178]MSQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV[GGGNGSGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI X; (7) [SEQ ID NO: 179]MSQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV[GGGCGGGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI X; (8) [SEQ ID NO: 180]MSQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV[GPNGG]_(n) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI X; and (9) [SEQ ID NO: 181]MSQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQVAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGES GEIKAIGELDLLFMSLRNACIX, wherein: MSQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV [SEQ ID NO: 46] =  α-endosulfine;Underlined text is flexible linker;Text in italics is IL-22 mature polypeptide;n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8,  9 or 10; andX is an optional tag (e.g., FLAGS, His6) for  purification, or (10)[SEQ ID NO: 182] MXHAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNA CI[GGGGSGGGGSGGGGSGGGG]_(n) SQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV; (11) [SEQ ID NO: 183] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNAC I [GGGSGGGS]_(n)SQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV; (12) [SEQ ID NO: 184] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNAC I[GGGG]_(n)SQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV; (13) [SEQ ID NO: 185] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNAC I [GGGGG]_(n)SQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV; (14) [SEQ ID NO: 186] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNAC I [GGGKGGGG]_(n)SQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV; (15) [SEQ ID NO: 187] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNAC I [GGGNGSGG]_(n)SQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV; (16) [SEQ ID NO: 188] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNAC I [GGGCGGGG]_(n)SQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV; (17) [SEQ ID NO: 189] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNAC I [GPNGG]_(n)SQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV; and (18) [SEQ ID NO: 190] MXAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACISQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV, wherein:SQKQEEENPAEETGEEKQDTQEKEGILPERAEEAKLKAKYPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNKQLPSAGPDKNLVTGDHIPTPQDLPQRKSSLVTSKLAGGQV [SEQ ID NO: 191] = α-endosulfine without initial methionine;Underlined text is flexible linker;Text in italics is IL-22 mature polypeptide;M is an artificial methionine for recombinant  expression;n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8,  9 or 10; andX is an optional tag (e.g., FLAGS, His6) for  purification.

In other embodiments, the OER stress inhibitor is part of a solublereceptor that binds to IL-24 and the targeting agent is anantigen-binding molecule that is immuno-interactive with a β-cellsurface binding protein as taught for example in WO 2010/096930.Non-limiting examples of constructs of this type include:

(A) [SEQ ID NO: 212] MXVPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [GGGGSGGGGSGGGGSGGGGS]_(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; (B) [SEQ ID NO: 213] MXVPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [GGGSGGGS]_(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; (C) [SEQ ID NO: 214] MXVPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [GGGG]_(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAE QKLISEEDLN;(D) [SEQ ID NO: 215] MXVPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [GGGGG]_(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAA EQKLISEEDLN;(E) [SEQ ID NO: 216] MXVPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [GGGKGGGG]_(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; (F) [SEQ ID NO: 217] MXVPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [GGGNGSGG]_(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; (G) [SEQ ID NO: 218] MXVPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [GGGCGGGG]_(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; (H) [SEQ ID NO: 219] MXVPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [GPNGG]_(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAA EQKLISEEDLN;(I) [SEQ ID NO: 220] MXVPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEE DLN; wherein:AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN [SEQ ID NO: 57] = β-cellspecific scFv Underlined text is flexible linker;Text in italics corresponds to an extracellular domain of IL-20RA fused to the heavy chain of IgGγ1;n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;X is an optional tag (e.g., FLAGS, His6) for purification, andwherein the construct is contacted with anextracellular domain of IL-20RB fused to the human κ lightchain, which comprises the sequence:DEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 199] to form a heterodimerthat binds IL-24, or (J) [SEQ ID NO: 221]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGGSGGGGSGGGGSGGGGS]_(n)VPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; (K) [SEQ ID NO: 222]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGSGGGS]_(n) VPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; (L) [SEQ ID NO: 223]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGG]_(n) VPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK;(M) [SEQ ID NO: 224]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGGG]_(n) VPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK;(N) [SEQ ID NO: 225]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGKGGGG]_(n) VPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; (O) [SEQ ID NO: 226]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGNGSGG]_(n) VPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; (P) [SEQ ID NO: 227]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGCGGGG]_(n) VPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; (Q) [SEQ ID NO: 228]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GPNGG]_(n) VPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK;(R) [SEQ ID NO: 229]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLNVPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK; wherein:AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN [SEQ ID NO: 57] = β-cellspecific scFv Underlined text is flexible linker;Text in italics corresponds to an extracellular domain ofIL-20RA fused to the heavy chain of IgGγ1;n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;X is an optional tag (e.g., FLAGS, His6) for purification, andwherein the construct is contacted with anextracellular domain of IL-20RB fused to the human κ lightchain, which comprises the sequence:DEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 199] to form a heterodimerthat binds IL-24, or (S) [SEQ ID NO: 230] MXDEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [GGGGSGGGGSGGGGSGGGGS]_(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; (T) [SEQ ID NO: 231] MXDEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [GGGSGGGS]_(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLI SEEDLN; (U)[SEQ ID NO: 232] MXDEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [GGGG]_(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEED LN; (V)[SEQ ID NO: 233] MXDEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [GGGGG]_(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEE DLN; (W)[SEQ ID NO: 234] MXDEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [GGGKGGGG]_(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLI SEEDLN; (X)[SEQ ID NO: 235] MXDEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [GGGNGSGG]_(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLI SEEDLN; (Y)[SEQ ID NO: 236] MXDEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [GGGCGGGG]_(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLI SEEDLN; (Z)[SEQ ID NO: 237] MXDEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [GPNGG]_(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEE DLN; (AA)[SEQ ID NO: 238] MXDEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN; wherein:AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN [SEQ ID NO: 57] = β-cellspecific scFv Underlined text is flexible linker;Text in italics corresponds to an extracellular domain ofIL-20RB fused to the human κ light chain;n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;X is an optional tag (e.g., FLAGS, His6) for purification, andwherein the construct is contacted with anextracellular domain of IL-20RA fusedto the heavy chain of IgGγ1, which comprises the sequence:VPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [SEQ ID NO: 198] to form a heterodimer that binds IL-24, or (AB) [SEQ ID NO: 239]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGGSGGGGSGGGGSGGGGS]_(n)DEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; (AC) [SEQ ID NO: 240]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGSGGGS]_(n) DEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC; (AD)[SEQ ID NO: 241]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGG]_(n) DEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC; (AE)[SEQ ID NO: 242]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGGG]_(n) DEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC; (AF)[SEQ ID NO: 243]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGKGGGG]_(n) DEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC; (AG)[SEQ ID NO: 244]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGNGSGG]_(n) DEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC; (AH)[SEQ ID NO: 245]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GGGCGGGG]_(n) DEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC; (AI)[SEQ ID NO: 246]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[GPNGG]_(n) DEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC; (AJ)[SEQ ID NO: 247]MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLNDEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLTEGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIELEDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAFSQTECVEVQGEATVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; wherein:AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN [SEQ ID NO: 57] = β-cellspecific scFv Underlined text is flexible linker;Text in italics corresponds to an extracellular domain of IL-20RB fused to the human κ light chain;n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;X is an optional tag (e.g., FLAGS, His6) for purification, andwherein the construct is contacted with anextracellular domain of IL-20RA fused to the heavy chainof IgGγ1, which comprises the sequence:VPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [SEQ ID NO: 198] to form a heterodimer that binds IL-24.

In still other embodiments, the OER stress inhibitor is anantigen-binding molecule that is immuno-interactive with IL-23 and thetargeting agent is an antigen-binding molecule that isimmuno-interactive with a β-cell surface binding protein as taught forexample in WO 2010/096930. Non-limiting examples of constructs of thistype include:

[SEQ ID NO: 248] (A) MXQSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLTVLGGGGSGGGG SGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNE YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAFDIWGQGTMVTVSS [GGGGSGGGGSGGGGSGGGGS] _(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR QAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFD YWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWY QQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTK VEIKRAAAHHHHHHGAAEQKLISEEDLN;  [SEQ ID NO: 249] (B) MXQSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLTVLGGGGSGGGG SGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNE YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAFDIWGQGTMVTVSS [GGGSGGGS] _(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN;  [SEQ ID NO: 250] (C) MXQSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLTVLGGGGSGGGG SGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNE YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAFDIWGQGTMVTVSS [GGGG] _(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGT HTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGS GGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQ SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAE QKLISEEDLN;  [SEQ ID NO: 251] (D) MXQSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLTVLGGGGSGGGG SGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNE YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAFDIWGQGTMVTVSS [GGGGG] _(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEG THTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGG SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN;  [SEQ ID NO: 252] (E) MXQSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLTVLGGGGSGGGG SGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNE YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAFDIWGQGTMVTVSS [GGGKGGGG] _(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN;  [SEQ ID NO: 253] (F) MXQSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLTVLGGGGSGGGG SGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNE YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAFDIWGQGTMVTVSS [GGGNGSGG] _(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN;  [SEQ ID NO: 254] (G) MXQSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLTVLGGGGSGGGG SGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNE YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAFDIWGQGTMVTVSS [GGGCGGGG] _(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN;  [SEQ ID NO: 255] (H) MXQSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLTVLGGGGSGGGG SGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNE YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAFDIWGQGTMVTVSS [GPNGG] _(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEG THTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGG SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN;  [SEQ ID NO: 256] (I) MXQSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNR PSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLTVLGGGGSGGGGS GGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNEY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAFDIWGQGTMVTVSSAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGG GGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDL  N; wherein:  AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTH TWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSG GGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQ KLISEEDLN;  [SEQ ID NO: 57] = β-cell specific scFv Underlined text is flexible linker;  Text in italics is anti-IL23 scFv; n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;  and X is an optional tag (e.g., FLAGS, His6) for purification,  or [SEQ ID NO: 257](J) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN[GGGGSGGGGSGGGGSGGGGS] _(n) QSVLTQPPSVSGAPGQRVTISCTGSSSNTG AGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSS LSGWVFGGGTRLTVLGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARD RGYTSSWYPDAFDIWGQGTMVTVSS; [SEQ ID NO: 258](K) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN[GGGSGGGS] _(n) QSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLT VLGGGGSGGGGSGGGGSQVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE WVAVIWYDGSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAF DIWGQGTMVTVSS;  [SEQ ID NO: 259](L) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN[GGGG] _(n) QSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLTVLG GGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAFDIWG QGTMVTVSS;  [SEQ ID NO: 260](M) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN[GGGGG] _(n) QSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLTVLG GGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAFDIWG QGTMVTVSS;  [SEQ ID NO: 261](N) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN[GGGKGGGG] _(n) QSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLT VLGGGGSGGGGSGGGGSQVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE WVAVIWYDGSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAF DIWGQGTMVTVSS;  [SEQ ID NO: 262](O) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN[GGGNGSGG] _(n) QSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLT VLGGGGSGGGGSGGGGSQVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE WVAVIWYDGSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAF DIWGQGTMVTVSS;  [SEQ ID NO: 263](P) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN[GGGCGGGG] _(n) QSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLT VLGGGGSGGGGSGGGGSQVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE WVAVIWYDGSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAF DIWGQGTMVTVSS;  [SEQ ID NO: 264](Q) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN[GPNGG] _(n) QSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLTVLG GGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAFDIWG QGTMVTVSS;  [SEQ ID NO: 265](R) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLNQSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSG NRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLTVLGGGGSGGG GSGGGGSQVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAFDIWGQGTMVTVS  S; wherein:  AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTH TWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSG GGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQ KLISEEDLN;  [SEQ ID NO: 57]= β-cell specific scFv Underlined text is flexible linker;  Text in italics is anti-IL23 scFv; n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;  and X is an optional tag (e.g., FLAGS, His6) for purification. 

In still other embodiments, the OER stress inhibitor is anantigen-binding molecule that is immuno-interactive with IL-23R and thetargeting agent is an antigen-binding molecule that isimmuno-interactive with a β-cell surface binding protein as taught forexample in WO 2010/096930. Non-limiting examples of constructs of thistype include:

[SEQ ID NO: 266] (a) MXDIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGG SQVQLVESGGGVVQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADINSKSYNYATYYAD SVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTVSS [GGGGSGGGG SGGGGSGGGGS] _(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVS SITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVS SGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI YKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHH HHHGAAEQKLISEEDLN;  [SEQ ID NO: 267] (b) MXDIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGG SQVQLVESGGGVVQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADINSKSYNYATYYAD SVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTVSS [GGGSGGGS] _(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSG GGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEE DLN;  [SEQ ID NO: 268] (c) MXDIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQDG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGGS QVQLVESGGGVVQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADINSKSYNYATYYADS VKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTVSS [GGGG] _(n)AMAEV QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGST DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[SEQ ID NO: 269] (d) MXDIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGG SQVQLVESGGGVVQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADINSKSYNYATYYAD SVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTVSS [GGGGG] _(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGG STDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[SEQ ID NO: 270] (e) MXDIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGG SQVQLVESGGGVVQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADINSKSYNYATYYAD SVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTVSS [GGGKGGGG] _(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSG GGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEE DLN;  [SEQ ID NO: 271] (f) MXDIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQDG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGGS QVQLVESGGGVVQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADINSKSYNYATYYADS VKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTVSS [GGGNGSGG] _(n)AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGG GGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDL N; [SEQ ID NO: 272] (g) MXDIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGG SQVQLVESGGGVVQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADINSKSYNYATYYAD SVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTVSS [GGGCGGGG] _(n) AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSG GGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEE DLN;  [SEQ ID NO: 273] (h) MXDIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGG SQVQLVESGGGVVQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADINSKSYNYATYYAD SVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTVSS [GPNGG]_(n)AMAE VQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKG RFTISRD NSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGS TDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN[SEQ ID NO: 274] (i) MXDIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQDG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGGS QVQLVESGGGVVQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADINSKSYNYATYYADS VKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTVSSAMAEVQLLESGG GLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQ SPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLN;  wherein: AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTH TWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSG GGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQ KLISEEDLN;  [SEQ ID NO: 57] = β-cell specific scFv Underlined text is flexible linker; Text in italics is anti-IL23R scFv; n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;  and X is an optional tag (e.g., FLAGS, His6) for purification,  or [SEQ ID NO: 275](j) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN[GGGGSGGGGSGGGGSGGGGS] _(n) DIQMTQSPSSLSASVGDRVTITCLASEDIY NNLAWYQQKPGKAPKLLIYHASSLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFDFNSYGMSWVR QAPGKGLEWVADINSKSYNYATYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSD YFEYWGQGTLVTVSS;  [SEQ ID NO: 276](k) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN[GGGSGGGS] _(n) DIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGWQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADI NSKSYNYATYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTV  SS; [SEQ ID NO: 277](l) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN[GGGG] _(n)DIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGWQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADINSKSY NYATYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTVSS; [SEQ ID NO: 278](m) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN[GGGGG] _(n)DIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADINSKS YNYATYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTVSS; [SEQ ID NO: 279](n) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN[GGGKGGGG] _(n) DIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGWQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADI NSKSYNYATYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTV  SS; [SEQ ID NO: 280](o) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN[GGGNGSGG] _(n) DIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGWQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADI NSKSYNYATYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTV  SS; [SEQ ID NO: 281](p) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN[GGGCGGGG] _(n) DIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGWQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADI NSKSYNYATYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTV  SS; [SEQ ID NO: 282](q) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLN[GPNGG] _(n)DIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADINSKS YNYATYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTVSS; [SEQ ID NO: 283](r) MXAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIT AEGTHTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKAS RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHG AAEQKLISEEDLNDIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRGGGGSGGGGSGGG GSQVQLVESGGGVVQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADINSKSYNYATYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTVSS;  wherein: AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITAEGTH TWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSYRFDYWGQGTLVTVSSGGGGSG GGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASRLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKWDPPRTFGQGTKVEIKRAAAHHHHHHGAAEQ KLISEEDLN;  [SEQ ID NO: 57] = β-cell specific scFv Underlined text is flexible linker; Text in italics is anti-IL23R scFv; n = 1 or more, suitably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;  and X is an optional tag (e.g., FLAGS, His6) for purification. 

In other embodiments of the anti-IL-24 and IL-23 constructs definedabove, the chimeric constructs comprise alternative β=cell specific scFvsubstituted for the scFv defined above, illustrative examples of whichinclude:

[SEQ ID NO: 58] AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSRIKIFGSKTKFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHSTHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLQSTPRTFGQGTKVEIKRAAAHHHH HHGAAEQKLISEEDLN; [SEQ ID NO: 59] AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIHPKGYPTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTTPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQMGRDPRTFGQGTKVEIKRAAAHHHH HHGAAEQKLISEEDLN; [SEQ ID NO: 60] AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSRIQFFGSHTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHSTHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNRRIPRTFGQGTKVEIKRAAAHHHHH HGAAEQKLISEEDLN; and  [SEQ ID NO: 61] AMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSTGDSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAADSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTNGAPTTFGQGTKVEIKRAAAHHHH HHGAAEQKLISEEDLN. 

6. Delivery Vehicle Embodiments

The present invention contemplates the use of delivery vehicles fortargeted delivery of an OER stress inhibitor to a β-cell. The deliveryvehicle will generally comprise a β-cell targeting agent and an OERstress inhibitor.

In some embodiments, the delivery vehicle is a polymeric particle.Polymeric particles may be formed from any biocompatible and desirablybiodegradable polymer, copolymer, or blend. The polymers may be tailoredto optimize different characteristics of the particle including i)interactions between the bioactive agents (i.e., β-cell targeting agentand OER stress inhibitor) and target proteins or receptors on the β-celland the polymer to provide stabilization of the bioactive agents andretention of activity upon delivery; ii) surface characteristics andtargeting capabilities via chemical modification; and iii) particleporosity.

Surface eroding polymers such as polyanhydrides may be used to form theparticles. For example, polyanhydrides such aspoly[(p-carboxyphenoxy)-hexane anhydride] (PCPH) may be used.Biodegradable polyanhydrides are described in U.S. Pat. No. 4,857,311.

In other embodiments, bulk eroding polymers such as those based onpolyesters including poly(hydroxy acids) or poly(esters) can be used.For example, polyglycolic acid (PGA), polylactic acid (PLA), orcopolymers thereof may be used to form the particles. The polyester mayalso have a charged or functionalizable group, such as an amino acid. Inillustrative examples, particles with controlled release properties canbe formed of poly(D,L-lactic acid) and/or poly(D,L-lactic-co-glycolicacid) (“PLGA”).

Other polymers include poly(alkylcyanoacrylates), polyamides,polycarbonates, polyalkylenes such as polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly vinyl compounds such as polyvinyl alcohols,polyvinyl ethers, and polyvinyl esters, polymers of acrylic andmethacrylic acids, celluloses and other polysaccharides, and peptides orproteins, or copolymers or blends thereof. Polymers may be selected withor modified to have the appropriate stability and degradation rates invivo for different controlled drug delivery applications.

In some embodiments, particles are formed from functionalized polymerssuch as polyester graft copolymers, as described in Hrkach et al. (1995,Macromolecules, 28:4736-4739; and “Poly(L-Lactic acid-co-amino acid)Graft Copolymers: A Class of Functional Biodegradable Biomaterials” inHydrogels and Biodegradable Polymers for Bioapplications, ACS SymposiumSeries No. 627, Raphael M. Ottenbrite et al., Eds., American ChemicalSociety, Chapter 8, pp. 93-101, 1996.)

Materials other than biodegradable polymers may be used to form theparticles. Suitable materials include various non-biodegradable polymersand various excipients. The particles also may be formed of thebioactive agent(s) and surfactant alone.

Polymeric particles may be prepared using single and double emulsionsolvent evaporation, spray drying, solvent extraction, solventevaporation, phase separation, simple and complex coacervation,interfacial polymerization, and other methods well known to those ofordinary skill in the art. Particles may be made using methods formaking microspheres or microcapsules known in the art, provided that theconditions are optimized for forming particles with the desireddiameter.

Methods developed for making microspheres for delivery of bioactiveagents are described in the literature, for example, as described inDoubrow, M., Ed., “Microcapsules and Nanoparticles in Medicine andPharmacy,” CRC Press, Boca Raton, 1992. Methods also are described inMathiowitz and Langer (1987, 3. Controlled Release 5, 13-22); Mathiowitzet al. (1987, Reactive Polymers 6, 275-283); and Mathiowitz et al.(1988, 3. Appl. Polymer Sci. 35, 755-774) as well as in U.S. Pat. Nos.5,213,812, 5,417,986, 5,360,610, and 5,384,133. The selection of themethod depends on the polymer selection, the size, external morphology,and crystallinity that is desired, as described, for example, byMathiowitz et al. (1990, Scanning Microscopy 4: 329-340; 1992, J. Appl.Polymer Sci. 45, 125-134); and Benita et al. (1984, 3. Pharm. Sci. 73,1721-1724).

In solvent evaporation, described for example, in Mathiowitz et al.,(1990), Benita; and U.S. Pat. No. 4,272,398 to Jaffe, the polymer isdissolved in a volatile organic solvent, such as methylene chloride.Several different polymer concentrations can be used, for example,between 0.005 and 2.0 g/mL. The bioactive agent(s), either in solubleform or dispersed as fine particles, is (are) added to the polymersolution, and the mixture is suspended in an aqueous phase that containsa surface-active agent such as poly(vinyl alcohol). The aqueous phasemay be, for example, a concentration of 1% poly(vinyl alcohol) w/v indistilled water. The resulting emulsion is stirred until most of theorganic solvent evaporates, leaving solid microspheres, which may bewashed with water and dried overnight in a lyophilizer. Microsphereswith different sizes (between 0.1 and 1000 μm) and morphologies can beobtained by this method.

Solvent removal was primarily designed for use with less stablepolymers, such as the polyanhydrides. In this method, the agent isdispersed or dissolved in a solution of a selected polymer in a volatileorganic solvent like methylene chloride. The mixture is then suspendedin oil, such as silicon oil, by stirring, to form an emulsion. Within 24hours, the solvent diffuses into the oil phase and the emulsion dropletsharden into solid polymer microspheres. Unlike the hot-meltmicroencapsulation method described for example in Mathiowitz et al.(1987, Reactive Polymers, 6:275), this method can be used to makemicrospheres from polymers with high melting points and a wide range ofmolecular weights. Microspheres having a diameter for example betweenone and 300 microns can be obtained with this procedure.

With some polymeric systems, polymeric particles prepared using a singleor double emulsion technique, vary in size depending on the size of thedroplets. If droplets in water-in-oil emulsions are not of a suitablysmall size to form particles with the desired size range, smallerdroplets can be prepared, for example, by sonication or homogenation ofthe emulsion, or by the addition of surfactants.

If the particles prepared by any of the above methods have a size rangeoutside of the desired range, particles can be sized, for example, usinga sieve, and optionally further separated according to density usingtechniques known to those of skill in the art.

The polymeric particles can be prepared by spray drying. Methods ofspray drying, such as that disclosed in PCT WO 96/09814 by Sutton andJohnson, disclose the preparation of smooth, spherical microparticles ofa water-soluble material with at least 90% of the particles possessing amean size between 1 and 10 μm.

In some embodiments, the particles are liposomes. Liposomes can beproduced by standard methods such as those reported by Kim et al. (1983,Biochim. Biophys. Acta 728, 339-348); Liu et al. (1992, Biochim.Biophys. Acta 1104, 95-101); Lee et al. (1992, Biochim. Biophys. Acta.1103, 185-197), Brey et al. (U.S. Pat. Appl. Pub. 20020041861), Hass etal. (U.S. Pat. Appl. Pub. 20050232984), Kisak et al. (U.S. Pat. Appl.Pub. 20050260260) and Smyth-Templeton et al. (U.S. Pat. Appl. Pub.20060204566). Additionally, reference may be made to Copeland et al.(2005, Immunol. Cell Biol. 83: 95-105) who review lipid basedparticulate formulations for the delivery of antigen, and to Bramwell etal. (2005, Crit Rev Ther Drug Carrier Syst. 22(2):151-214; 2006, 3 PharmPharmacol. 58(6):717-728) who review particulate delivery systems forvaccines, including methods for the preparation of protein-loadedliposomes. Many liposome formulations using a variety of different lipidcomponents have been used in various in vitro cell culture and animalexperiments. Parameters have been identified that determine liposomalproperties and are reported in the literature, for example, by Lee etal. (1992, Biochim. Biophys. Acta. 1103, 185-197); Liu et al. (1992,Biochim. Biophys. Acta, 1104, 95-101); and Wang et al. (1989, Biochem.28, 9508-951).

In some embodiments, preparative methods based on hydration ofdried-lipid film are used, in which the lipids of choice (and anyorganic-soluble bioactive), dissolved in an organic solvent, are mixedand dried onto the bottom of a glass container under vacuum. The lipidfilm is rehydrated using an aqueous buffered solution containing anywater-soluble bioactives to be encapsulated by gentle swirling. Thehydrated lipid vesicles can then be further processed by extrusion,submitted to a series of freeze-thawing cycles or dehydrated and thenrehydrated to promote encapsulation of bioactives. Liposomes can then bewashed by centrifugation or loaded onto a size-exclusion column toremove unentrapped bioactive from the liposome formulation and stored at4° C. The basic method for liposome preparation is described in moredetail in Thierry et al. (1992, Nuc. Acids Res. 20:5691-5698).

In other embodiments, the delivery agent is a cyclodextrin.Cyclodextrins are cyclic oligosaccharides, which have a centralhydrophobic cavity and multiple hydroxyl groups on the outer surface.Bioactive molecules (e.g., β-cell targeting agent and OER stressinhibitor) can be conjugated to the cyclodextrin molecules through thesehydroxyl groups by methods known in the art. Cyclodextrins havetherefore already been used for the transport of anti-senseoligonucleotides in human T cell lines (Antisense Res Dev, 5:185-192,1995) and have also been used in vivo for intracellular transport andfor intracellular release or delivery of immunogenic CpG sequences(Biochem Pharmacol, 52:1537-1544, 1996). A wide variety of formulationsof cyclodextrins are given in the review article by Davis and Brewster(Nature Reviews Drug Discovery 3:1023-1035, 2004).

In still other embodiments, the delivery vehicle is a dendrimer.Dendrimers comprise a category of branched materials with diversefunctions that can be constructed with defined architectural andchemical structures. When decorated with bioactive molecules (e.g.,β-cell targeting agent and OER stress inhibitor) through peripheralchemical groups, dendrimer conjugates are can be turned intonanomaterials possessing binding properties with the cognate receptors.Numerous methods are known in the art for decorating dendrimers withbioactive molecules and reference may be made in this regard to Liu etal. (2012, Interface focus 2(3):307-24) for an illustrative review.

7. Compositions

In accordance with the present invention, the therapeutic agentsdescribed herein can improve pancreatic β-cell ER function, improveinsulin biosynthesis, increase glucose tolerance, modulate expression ofoxidative stress regulatory genes, reduce stress induced by lipids,glucose, inflammatory cytokines or environmental ROS, e.g., via STAT1-and STAT3-mediated upregulation of anti-oxidant genes and suppression ofoxidative stress-inducing genes, reduce ER stress, promote secretion ofhigh quality efficacious insulin, restore glucose homeostasis and/orenhancing peripheral insulin sensitivity. Accordingly, it is proposedthat these therapeutic agents will be useful for treating metabolicdisorders, including pre-diabetes, diabetes (type I or type II),metabolic syndrome, obesity, non-alcoholic fatty liver disease (NAFLD),non-alcoholic steatohepatitis (NASH), diabetic dyslipidemia,hyperlipidemia, hypertension, hypertriglyceridemia, hyperfattyacidemia,and hyperinsulinemia.

The therapeutic agents described herein can be administered to a patienteither by themselves, or in pharmaceutical compositions where they aremixed with a suitable pharmaceutically acceptable carrier. The choice ofcarrier will generally depend on whether the therapeutic agents are tobe administered in solid, liquid or aerosol form, and whether they needto be sterile for such routes of administration as injection. Thetherapeutic agents can be administered intravenously, intradermally,transdermally, intrathecally, intraarterially, intraperitoneally,intranasally, intravaginally, intrarectally, topically, intramuscularly,subcutaneously, mucosally, orally, topically, locally, inhalation (e.g.,aerosol inhalation), injection, infusion, continuous infusion, localizedperfusion bathing target cells directly, via a catheter, via a lavage,in cremes, in lipid compositions (e.g., liposomes), or by other methodor any combination of the forgoing as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, incorporated herein by reference).In specific embodiments, the therapeutic agents are administeredsystemically, suitably by injection.

The therapeutic agents may be formulated into a composition in a freebase, neutral or salt form. Pharmaceutically acceptable salts, includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as formulated for parenteraladministrations such as injectable solutions, or aerosols for deliveryto the lungs, or formulated for alimentary administrations such as drugrelease capsules and the like.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the therapeutic agents are contained in aneffective amount to achieve their intended purpose. For any therapeuticagent used in the treatment methods of the present invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes the IC50 asdetermined in cell culture (e.g., the concentration of a test agent,which achieves a half-maximal inhibition of β-cell OER stress or aβ-cell OER stress-inducing polypeptide (e.g., IL-23, IL-24, etc.). Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of such drugs can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit large therapeutic indices are preferred. The dataobtained from these cell culture assays and animal studies can be usedin formulating a range of dosage for use in human. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED50 with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See for example Fingl et al., 1975, in“The Pharmacological Basis of Therapeutics”, Ch. 1 p1).

Generally, the dose of therapeutic agent administered to a patientshould be sufficient to achieve a beneficial response in the patientover time such as an improvement in pancreatic β-cell ER function,improvement in insulin biosynthesis, increase glucose tolerance,beneficial modulation of expression of oxidative stress regulatorygenes, reduction in stress induced by lipids, glucose, inflammatorycytokines or environmental ROS, e.g., via STAT1- and STAT3-mediatedupregulation of anti-oxidant genes and suppression of oxidativestress-inducing genes, reduction in ER stress, increase in secretion ofhigh quality efficacious insulin, restoration of glucose homeostasisand/or enhancing peripheral insulin sensitivity. The quantity of thedrug(s) to be administered may depend on the subject to be treatedinclusive of the age, sex, weight and general health condition thereof.In this regard, precise amounts of the drug(s) for administration willdepend on the judgment of the practitioner. In determining the effectiveamount of the drug to be administered in the modulation of type 2diabetes, the physician may evaluate tissue or cell levels of astress-modulating cytokine, glucose levels, HbA1c levels, insulinlevels, blood pressure, High Density Lipoprotein (HDL) levels,triglycerides levels, uric acid levels, weight loss etc. In any event,those of skill in the art may readily determine suitable dosages of thedrugs of the invention.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active agent that are sufficient to stimulate ormaintain inhibition or reduction of β-cell OER stress. Usual patientdosages for systemic administration range from 0.1-2000 mg/day, commonlyfrom 1-250 mg/day, and typically from 10-150 mg/day. Stated in terms ofpatient body weight, usual dosages range from 0.02-25 mg/kg/day,commonly from 0.02-3 mg/kg/day, typically from 0.2-1.5 mg/kg/day. Statedin terms of patient body surface areas, usual dosages range from0.5-1200 mg/m²/day, commonly from 0.5-150 mg/m²/day, typically from5-100 mg/m²/day.

In light of the abundance of IL-22 receptor on the cell surface of aβ-cell, in embodiments wherein the OER stress inhibitor is IL-22, thepharmaceutical composition is administered to a subject at aconcentration of between around 0.02 to around 0.1 mg/kg/day (1.25-6nmoles/kg/day). This is substantially below the usual doses of suchtherapeutics, and thus, the negative side effects associated with IL-22administration, or the unforeseen side effects of the targeting agent,would not appear at this low dosage.

In some embodiments, therapeutic efficacy is established or monitored bydetermining ER stress. In this regard, ER stress markers and methods formeasuring them have been previously identified and are known in the art(see, Iwawaki et al., 2004). For example, analysis of Xbp1 mRNAsplicing, ATF6 nuclear translocation, eIF2a phosphorylation, and Griesstest are all suitable assays for determining ER stress (see, Cunha etal., 2008). These ER stress markers may be measured using any techniquesknown in the art for measuring mRNA levels, protein levels, proteinactivity, or phosphorylation status. Exemplary techniques for measuringER stress markers include western blot analysis, ELISA, northern blotanalysis, immunoassays, quantitative PCR analysis, and enzyme activityassay (for a more detailed description of these techniques, please seeAusubel et al. Current Protocols in Molecular Biology (John Wiley &Sons, Inc., New York, 1999); Molecular Cloning: A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring HarborLaboratory Press: 1989); each of which is incorporated herein byreference). The levels of ER stress markers may be determined from anycells in the subject's body. In certain embodiments, the cells are cellsfrom a pancreatic β-cell. The cells may be obtained in any mannerincluding biopsy or surgical excision. Oxidative stress can be measuredby chemical assays assessing the presence of reactive oxygen or reactivenitrogen species such as, but not limited to, O₂ ⁻, H₂O₂, NO, OH andONOO⁻, or metabolites thereof. These assays can be applied to culturedβ-cells, cultured pancreatic islets or to tissue samples includinghistological sections. Another way of determining the tissue specificityof OER stress inhibitors incorporating, for example, IL-22 or IL-10 isto determine the relative changes in STAT1 and STAT3 phosphorylation inpancreatic β-cells versus other IL-22R1-expressing cell type in theintestinal epithelium, respiratory epithelium, cutaneous epithelium andliver. Successful targeting to the OER stress inhibitor will beaccompanied by an increase in the ratio of STAT1/3 phosphorylationbetween β-cells and the other IL-22R1-expressing cell types versusnative IL-22.

In following, for example, the progression of a disease associated withOER stress, or the effectiveness of present invention in therapy, onemay determine the levels of one, two, three, four, five, or six OERstress markers. In certain embodiments, the level of only one OER stressmarker is determined. In other embodiments, the levels of at least twoOER stress markers are determined. In yet other embodiments, the levelsof at least three OER stress markers are determined.

In some embodiments, the therapeutic agents of the present invention areadministered concurrently with one or more ancillary agents ortreatments that are known or approved for treating a metabolic disorder.For example, an individual that is obese may be administered acomposition of the invention in addition to another therapy for obesity.Additional obesity therapies include dietary therapy, physical therapy(exercise), drug therapy, surgery, and behavioral therapy, for example.Exemplary anti-obesity drug therapies include, for example, XenicalOrlistat®, phentermine, sibutramine (Meridia®), ATL-962, AJ9677,L750355, CP331648, topiramate, axokine, dexamphetamine,phenylpropanolamine, famoxin, or mazindol. Exemplary surgeries includeliposuction and gastric bypass, for example.

For individuals with diabetes, for example, exemplary additionalcompounds for therapy include one or more of the following: metformin,glyburide, glimepiride, glyburide, glipyride, glipizide, chlorpropamide,gliclazide, acarbose, miglitol, pioglitazone, troglitazone,dapagliflozin, rosiglitazone, sitagliptin, Avandaryl(rosiglitazone+glimepiride), Avandamet (rosiglitazone and metformin),Duetact (pioglitazone and glimepiride), Glucovance(glyburide/metformin), Metaglip (glipizide+metformin), insulin,GI-262570, isaglitazone, taspoglutide, albiglutide, JTT-501, NN-2344,L895645, YM-440, R-119702, A39677, repaglinide, nateglinide, KAD1129,APR-H039242, GW-409544, KRP297, AC2993, Exendin-4, liraglutide,LY307161, NN2211 or LY315902, Vildagliptin, glucosidase inhibitor,sodium-glucose co-transporter 2 (SGLT2) inhibitors such as canaglifozinand pramlintide. Other therapies for diabetes include an improvement indiet and exercise.

For individuals with high triglycerides or fatty acid levels, exemplaryadditional compounds for therapy include one or more lipid-modulatingagents such as but not limited to pravastatin, lovastatin, simvastatin,atorvastatin, cerivastatin, fluvastatin, nisvastatin, ZD-4522,fenofibrate, gemfibrozil, clofibrate, implitapide, CP-529,414,avasimibe, TS-962, MD-700, or LY295427.

8. Therapeutic Uses

The present invention provides methods for improving pancreatic β-cellER function, improving insulin biosynthesis, increasing glucosetolerance, modulating expression of oxidative stress regulatory genes,reducing stress induced by lipids, glucose, inflammatory cytokines orenvironmental ROS, e.g., via STAT1- and STAT3-mediated upregulation ofanti-oxidant genes and/or suppression of oxidative stress-inducinggenes, reducing ER stress, promoting secretion of high qualityefficacious insulin, restoring glucose homeostasis and/or enhancingperipheral insulin sensitivity.

Thus, in some embodiments, the invention provides methods for improvingpancreatic β-cell ER function in a β-cell, comprising contacting theβ-cell with a pancreatic β-cell ER function-improving amount of atherapeutic agent as broadly described above and elsewhere herein.

In some embodiments, the invention provides methods for improvinginsulin biosynthesis in a β-cell, comprising contacting the β-cell withan insulin biosynthesis-improving amount of a therapeutic agent asbroadly described above and elsewhere herein.

In some embodiments, the invention provides methods for modulatingexpression of oxidative stress regulatory genes, comprising contactingthe β-cell with an oxidative stress regulatory geneexpression-modulating amount of a therapeutic agent as broadly describedabove and elsewhere herein.

In some embodiments, the invention provides methods for reducing stressinduced by lipids, glucose, inflammatory cytokines or environmental ROS,e.g., via STAT1- and STAT3-mediated upregulation of anti-oxidant genesand/or suppression of oxidative stress-inducing genes in a β-cell,comprising contacting the β-cell with an amount of a therapeutic agentas broadly described above and elsewhere herein, which is effective forreducing that stress.

In some embodiments, the invention provides methods for reducing ERstress in a β-cell, comprising contacting the β-cell with anER-stress-reducing amount of a therapeutic agent as broadly describedabove and elsewhere herein.

In some embodiments, the invention provides methods for increasingglucose tolerance in a subject, comprising administering to the subjecta glucose tolerance-increasing amount of a therapeutic agent as broadlydescribed above and elsewhere herein.

In some embodiments, the invention provides methods for promotingsecretion of high quality insulin from a β-cell, comprising contactingthe β-cell with an amount of a therapeutic agent as broadly describedabove and elsewhere herein, which is effective for promoting thatsecretion.

In some embodiments, the invention provides methods for increasingglucose tolerance in a subject, comprising administering to the subjecta glucose tolerance-increasing amount of a therapeutic agent as broadlydescribed above and elsewhere herein.

In some embodiments, the invention provides methods for restoringglucose homeostasis in a subject, comprising administering to thesubject a glucose homeostasis-restoring amount of a therapeutic agent asbroadly described above and elsewhere herein.

In some embodiments, the invention provides methods for enhancinginsulin sensitivity in a subject, comprising administering to thesubject an insulin sensitivity-enhancing amount of a therapeutic agentas broadly described above and elsewhere herein.

In some embodiments, the invention provides methods for stimulating orenhancing weight loss in a subject, comprising administering to thesubject a weight loss-stimulating or -enhancing amount of a therapeuticagent as broadly described above and elsewhere herein.

In accordance with the present invention, it is proposed that thesetherapeutic agents will be useful for treating metabolic disorders,including pre-diabetes, diabetes (type I or type II), metabolicsyndrome, obesity, non-alcoholic fatty liver disease (NAFLD),non-alcoholic steatohepatitis (NASH), diabetic dyslipidemia,hyperlipidemia, hypertension, hypertriglyceridemia, hyperfattyacidemia,and hyperinsulinemia. Accordingly, the present invention providesmethods for treating a metabolic disorder in a subject, comprisingadministering to the subject an effective amount of a therapeutic agentas broadly described above and elsewhere herein.

In particular aspects of the invention, an individual is given one ormore compositions of the present invention and the individual isassessed for an improvement in at least one symptom of the metabolicdisorder. For example, in particular embodiments when the metabolicdisorder is obesity, an improvement in obesity may be determined duringand/or following treatment with one or more compositions of theinvention. An improvement in obesity may be measured by any standardmeans, but in particular aspects the improvement in obesity is measuredby weight measurement, body mass index (BMI) measurement, and/or bodypart size measurement (such as waist measurement), for example.Exemplary methods for calculating BMI includes dividing a person's bodyweight in kilograms by their height in meters squared (weight [kg]height [m]²). For example, for Caucasians, a BMI of 30 or more isconsidered obese and a BMI between 25 and 29.9 is considered overweight.

In other aspects of the invention, an individual with diabetes is testedfor an improvement following administration to the individual of thetherapy of the invention. In one specific embodiment, the monitoring ofdiabetes occurs by blood test. For example, the blood test may measurethe chemical A1C. The higher the blood sugar, the higher the A1C levelwill be. The American Diabetic Association recommends that diabeticsmaintain a A1C of less than 7.0% to reduce the complications associatedwith diabetes. (The American Association of Clinical Endocrinologistsrecommend 6.5% or less).

In some cases, cholesterol (including HDL and/or LDL cholesterol) and/ortriglycerides are measured, such as by standard means in the art. Inspecific cases, a fasting lipoprotein profile is performed, such as bystandard means in the art.

In order that the invention may be readily understood and put intopractical effect, particular preferred embodiments will now be describedby way of the following non-limiting examples

Example 1 Identification of Cytokines that Drive ER Stress

β-cell specific deficiency of a critical Unfolded Protein Response (UPR)transcription factor, Xbp1, decreases insulin secretion, impairsproinsulin processing and increases serum proinsulin, resulting inhyperglycemia and glucose intolerance, phenocopying T2D. It wastherefore hypothesized that inflammation-induced ER stress underpinsβ-cell dysfunction in T2D and impairs the quality and quantity ofsecreted insulin. Protein misfolding activates the IRE-1endoribonuclease that specifically splices Xbp1 mRNA, and thereforeXbp1-splicing is a direct measure of the UPR and ER stress.

Screening a panel of proinflammatory cytokines using the ERAI reporterof Xbp1-splicing transfected into murine MIN6N8 β-cells, identifiedseveral cytokines as inducing ER stress. Specifically, IL-23, IL-24 andIL-33 were determined to be potent inducers of ER stress, with a lowerstress response induced by IL-1β, MIP-2α, IL-17A and IFN-γ and IFN-β(FIG. 1A).

It was shown using pharmacological inhibitors that each of thesecytokines induces ER stress by distinct mechanisms. IL-23 acts viaSTAT3, IL-24 via STAT1 and IL-33 via STAT1 and NFκB. All of the ERstress-inducing cytokines trigger production of reactive oxygen species(ROS) and/or nitric oxide (NO) (FIG. 1C). Pharmacological inhibitorsshowed the various cytokines induced ER stress via oxidative stress withalmost complete inhibition of ER stress with a combination of GSH, SOD1and L-NMMA. ER stress was generated by downstream activation of a rangeof transcription factors, for example, IL-23 via STAT3, IL-24 via STAT1,and IL-33 requires both STAT5 and NFκB (see, Table 3, below). Thusmultiple cytokines induce classical ER stress in β-cells by induction ofoxidative stress involving both reactive oxygen and reactive nitrogenspecies.

TABLE 3 RG + SOD1 + Required Excluded GSH SOD1 L-NMMA L-NMMATranscription Transcription Cytokine % Inhibition of ER Stress (Xbp1splicing) Factors/Kinases Factors/Kinases IFNγ 15 74 87 98 NFκB STAT1IL-1β 16 96 93 100 NFκB ERK, JNK IL-17A 0 65 83 95 NFκB, JNK¹ ERK IL-2387 95 78 96 STAT3 STAT1, STAT5 IL-24 6 59 92 100 STAT1 STAT3 IL-33 13 7490 96 NFκB, STAT5² ERK, JNK, STAT1, STAT3 MIP-2α 7 88 95 97 Noneidentified ERK, JNK, NFκB, STAT1, STAT3, STAT5 GSH = reduced glutathione(scavenger of oxygen radicals); SOD1 = superoxide dismutase (enzyme thatdeactivates OH⁻); L-NMMA = N^(G)-Methyl-L-arginine (nitric oxideinhibitor). Note that the combination of inhibitors almost completelyprevents cytokine-induced ER stress. Transcription factors wereinhibited with both pharmacological inhibitors and siRNA to determinewhich of the known transcription factors downstream of each cytokinewere required to induce ER stress. ¹IL-17A can induce ER stress byeither NFκB or JNK, but more potently when both transcription factorsare involved. ²IL-33 induction of ER stress requires both NFκB andSTAT5.

Although these cytokines have not previously been considered to beinvolved in pancreatic pathophysiology in T2D, expression of all of theER stressor cytokine genes is increased in the pancreas of obese mice ona high fat diet (HFD), as shown in FIG. 2. IL-24 and IL-33 are amongstthe 20 most highly upregulated genes in human type 2 diabetes, showingrelevance for the human disease.

Increased pancreatic ER stress in obese diabetic mice coincides withincreased serum proinsulin, as shown in FIGS. 1E-H. Localization of theER stress chaperone, Grp78, showed that most ER stress occurs in thehighly secretory β- and α-cells (see, FIG. 1I), and accompanied by adecrease in β-cell insulin content. Additionally, a relocation of someα-cells from the periphery to the center of the islets was observed.

Materials and Methods In Vitro Assessments of ER/Oxidative Stress

MIN6N8 cells are maintained in line with standard protocols. Cells aretransfected with either the ERAI reporter plasmid (F-XBP1ΔDBD-Venus), orthe ATF6-GFP plasmid (ADGENE), using LIPFECTAMINE 2000 according to themanufacturer's instructions.

MIN6N8 cells comprising the ERAI reporter plasmid exhibit negligiblefluorescence in the absence of an ER stressor. On induction of ERstress, splicing of XBP2 mRNA by IRE1a results in the translation ofVenus, but not an active form of XBP1. Fluorescence was measured(excitation: 485 nm; emission: 520 nm) using the POLARstar Omega platereader and cells were treated 48 h after transfection. The results arenormalized with Gapdh expression as fold of vehicle-treated control(Con) cells.

Activation of ATF6 was assessed as follows: the area in pixels persquare millimeter co-stained ATF6 and DAPI (blue and green), and totalATF6 staining (green) was determined in 5 fields of view for eachsection (n=6 for each condition) using IMAGEJ software version 1.45s.Data are presented as the percentage of ATF6 and DAPI co-fluorescence tothe total ATF6 fluorescence. Phosphorylation of Ser-51 of eiF2 a wasdetermined by an AlphaScreen assay as per the manufacturer'sinstructions (PERKIN-ELMER). The Griess reagent method was performedaccording to the manufacturer's instructions (MOLECULAR PROBES) toassess the intracellular levels of nitric oxide.5-(and-6-)-carboxy-2,7-dichlorofluorescein diacetate (DCFDA) assay wasused to determine the intracellular levels of H₂O₂ as previouslydescribed (Sakon et al., 2003). Cells were treated with glucose orrecombinant cytokines at the concentrations indicated in the figurelegends, or with 5 μM thapsigargin (SIGMA-ALDRICH) or 10 μg/mLtunicamycin (SIGMA-ALDRICH) or 0.1-0.5 mM Palmitate (SIGMA-ALDRICH) forthe times indicated in the legends. ROS was inhibited using thedihydroethidium (DHE) fluorescent cell-permeable probe (SIGMA-ALDRICH).MIN6N8 cells plated at 2×10⁶/mL were incubated with 3 μM DHE andsubsequently treated with DMSO (control), 50 ng/mL IL-23 for 30minutes±50 mg/mL IL-22 at the same time or 30 minutes prior to IL-23.

To inhibit transcription factors the following specific inhibitors (andconcentrations) were used:

TABLE 4 Transcription Factor Inhibitor Concentration (μM) STAT1Fludarabine (MERK) 50 STAT3 VI S31-201 (SANTA CRUZ) 50 STAT5 573108(MERK) 50 JNK Inhibitor V (MERK) 10 ERK FR18020 (MERK) 5 NFκB BAY11-7085(CELL SIGNALING) 20

Cytokine Assay to Determine Impact on Er Stress

MIN6N8 cells were exposed to a panel of cytokines to determine theeffect of each cytokine on ER stress. ER stress was measured using theabove-described method. Specifically, the cells were exposed to either a24 hour treatment of any one of 1.6 mM glucose (hi glucose), 10 μM H₂O₂,0.5 mM palmitic acid or 50 ng/mL cytokines; or a 6 hour treatment with10 μg/mL tunicamycin or 5 μM thapsigargin. Tunicamycin is removed after6 hours as prolonged treatment induced cell death.

Cytokine Assay to Determine Impact on Er Stress

MIN6N8 cells were exposed to a panel of cytokines to determine theeffect of each cytokine on ER stress. ER stress was measured using theabove-described method. Specifically, the cells were exposed to either a24 hour treatment of any one of 1.6 mM glucose (hi glucose), 10 μM H₂O₂,0.5 mM palmitic acid or 50 ng/mL cytokines; or a 6 hour treatment with10 μg/mL tunicamycin or 5 μM thapsigargin. Tunicamycin is removed after6 hours as prolonged treatment induced cell death.

Quantitative Real Time-PCR (QRT-PCR)

Tissue samples were snap-frozen, and homogenized in TRIZOL usingFASTPREP (MP BIOMEDICAL). RNA was isolated using High Pure RNA isolationkit (ROCHE), followed by cDNA synthesis using iScript (BIORAD)containing oligo(dT) and random hexamers. QPCR SYBR Green (INVITROGEN)was used for quantitative PCR using a HT7900 (ABI SYSTEMS). RoX was usedas a normalization reference. Results were analyzed using SDS software(version 2.3) (ABI SYSTEMS). Primer efficiencies were determined usingcDNA dilutions and primer dilutions for the genes of interest. All thedata was normalized against the housekeeping gene, Gapdh, and expressedas a fold difference to the mean of relevant control samples.

Primers for PCR analysis were as follows:

sXBP-1:  [SEQ ID NO: 192] forward-5′ CTGAGTCCGCAGCAGGTG 3′; [SEQ ID NO: 193] reverse-5′ TGCCCAACAGGATATCAGACT 3′;  GRP78: [SEQ ID NO: 194] forward-5′ CACAGTGGTGCCTACCAAGA3′;  [SEQ ID NO: 195]reverse-5′ TGATTGTCTTTTGTCAGGGGT 3′. 

Oxidative Stress Array: cDNA from MIN6N8 cells treated with DMSO or 50ng/mL IL-22 was compared using the oxidative stress RT² Profiler PCRArray (QIAGEN, PAMM-065Z). Data were analyzed using RT² Profiler PCRArray data analysis software.

Quantification of Gene Expression

Total RNA was extracted from animal tissues by homogenizing in TRIzoland separated via chloroform phase separation. RNA was purified usingHigh Pure RNA isolation kit (ROCHE) as per the manufacturer'sinstructions, including DNASE treatment. Extraction purity was assessedby NANODROP, ensuring the expected A260/280 value for RNA between 1.8and 2.0. Reverse transcription was performed on 1 μg of RNA using5×ISCRIPT reaction mix and ISCRIPT reverse transcriptase (BIORAD).Quantitative, reverse transcription polymerase chain reaction (qRT-PCR)was performed to quantify expression of PVM gene Src homology (SH),using specific exon-spanning primer sequences and amplified with SYBRPremix Ex TAQ II polymerase (TAKARA). All gene expression was quantifiedin duplicate using a Viia 7 Real Time PCR System (APPLIED BIOSYSTEMS)and relative fold changes were calculated using the 2^(ΔΔCt) method,comparing to the mean of naïve uninfected mice after normalizing to theβ-actin housekeeping gene ACTB.

Statistical Analysis

In vitro experiments were powered to see a 2.0 standard deviation effectsize in a variety of primary outcome measures. Statistical analysis wasperformed using GRAPHPAD PRISM version 5.01 (GRAPHPAD SOFTWARE, INC).Differences between groups were assessed by using parametric tests afterconfirmation of a normal distribution by probability plots (one-wayANOVA with post-test or, where appropriate, a two-tailed Student t test,as described in individual figure legends).

Example 2 Identification of Cytokines that Inhibit Er Stress and/orOxidative Stress

Exposing β-cells to pair-wise combinations of cytokines revealed thatIL-22 and IL-10 inhibit ER stress, detected by the ERAI reporter genefor splicing of the Xbp1 mRNA, initiated by cytokines or theN-glycosylation inhibitor, tunicamycin (see, FIG. 2A). IL-22 is the mostpotent suppressor with efficacy as low as around 5 ng/mL in MIN6β-cells.

IL-22 also inhibits ER stress induced with the free fatty acid (FFA),palmitic acid (see, FIG. 2B), which induces β-cell oxidative and ERstress. IL-22 alone does not appear to activate the UPR, however, whenER stress is induced with thapsigargin, which disturbs ER Ca²⁺, IL-22increases UPR activation (see, FIG. 2C). Inhibitor of both STAT1 andSTAT3 transcription factors is required to block IL-22-mediatedsuppression or inhibitor of ER stress (see, FIG. 2D).

The pancreas has the highest tissue expression of the IL-22 receptor(IL-22R1), particularly in β- and α-cells, and IL-22 inhibition ofβ-cell ER stress is completely inhibited by an IL-22R1 antibody (see,FIG. 2D).

It was hypothesized that IL-22 prevented cytokine-induced ER stress byinhibiting oxidative stress. IL-23 induced oxidation of the fluorescentdye dihydroethidium (DHE), which is activated by 02, progressively over30 minutes and concomitant exposure to IL-22 reduced the degree ofoxidation and the number of cells affected. Notably, a 30-minutepre-exposure to IL-22 completely prevented DHE oxidation (see, FIG. 3A).IL-22 represses the production/accumulation of ROS and NO in β-cellsexposed to inflammatory cytokines, FFA, tunicamycin or H₂O₂, anddecreased basal NO production in unstressed cells (see, FIGS. 3B,C, andFIG. 4).

Given that IL-22 acts via transcription factors, the changes inexpression of oxidative stress pathway genes in non-stressed β-cellstreated with IL-22 were assessed using a RT-PCR array (see FIG. 5A). Theresults confirmed candidate genes by qRT-PCR (see, FIG. 5B). IL-22down-regulates three key oxidative stress inducing genes: Nos2 (nitricoxide synthase-2/iNos, catalyzes NO production), Hsp90ab1 (heat shockprotein involved in peroxynitrite production), and Fth1 (ferritin heavychain 1, a carrier of Fe³⁺).

Concomitantly, IL-22 upregulated the antioxidant genes Gpx5 (glutathioneperoxidase-5), Prdx5 (peroxiredoxin-5), and Sod2 (superoxidedismutase-2), the cyclooxygenases Ptgs1 and Ptgs2 (COX-1 and COX-2), andCyba (the p22^(phox) regulatory component of NADPH-oxidase). Thus IL-22regulated multiple genes involved in the oxidative stress pathwayexplaining its ability to suppress both oxidative and ER stress.

Materials and Methods

Experiments to assess ER and/or oxidative stress, and methods of qRT-PCTand/or quantifying gene expression were performed as described forExample 1. Xbp1 splicing is assessed by qRT-PCR, with normalizationversus Gapdh, and expression presented as fold of vehicle-treatedcontrol cells.

ER Stress Normalization by IL-22 Cotreatment or Pretreatment

To determine whether IL-22 was sufficient to prevent the increase inoxidative stress caused by known stressors, for example dihydroethidium(DHE), MIN6N8 cells were exposed to 50 ng/mL IL-23 for 30 minutes±50ng/mL IL-22 at the same time, or 30 minutes prior to the IL-23treatment. Superoxide generation was measured using DHE, whichfluoresces red and binds DNA after oxidation. Time-lapse imaging (3frames/min for 30 minutes) was utilized to determine the formation ofsuperoxide (DHE-conversion/fluorescence) in cells after treatment. DAPIfluorescence was captured to visualize nuclei.

The concentration of ROS and NO in MIN6N8 cells treated with 16.6 mMglucose (high glucose), 10 μM H₂O₂, 0.5 mM palmitic acid or 50 ng/mLcytokines±50 ng/mL IL-22 either at the same time, or 30 minutes prior tothe stressor treatment.

Example 3 Acute and Chronic ER Stress-Induced and Suppression of InsulinSecretion is Prevented by IL-22 Acute ER Stress

In healthy murine islets, treatment with tunicamycin, thapsigargin,IL-O, IL-23 and IL-24 induces Xbp1-splicing and Hspa5 mRNA (Grp78, ERchaperone upregulated during ER stress) (FIG. 6A). These ER stressagents decreased insulin secretion by islets in response to both glucoseand glucose plus GLP-1, while increasing inappropriate secretion ofproinsulin (FIGS. 6B,C). Co-treatment with IL-22 suppresses the acuteislet oxidative stress-driven ER stress and restores appropriate glucoseand glucose plus GLP-1 stimulated insulin secretion (FIGS. 6b ,C).

High Fat Diet Induced Obesity (HFDIO)

In healthy murine islets ex vivo treatment with IL-22R1 blockingantibody increased ER stress, decreased anti-oxidant gene expression,increased Nos2 (FIG. 7A), and decreased GSIS (FIG. 7B). Islets from micewith high fat diet-induced obesity (HFDIO) exhibited ER stress andupregulation of anti-oxidant genes decreased Nos2 (FIG. 7A). Culturewith IL-22 reduced ER stress, increased antioxidant gene expression anddecreased Nos2 (FIG. 7A).

In contrast to acute ER stress, HFDIO islets, which are adapted tochronic ER stress, showed increased GSIS. Insulin hyper-secretion wasaccentuated by culture in IL-22R1 antibody, and returned to levels innon-obese islets after culture in IL-22 (FIG. 7B). HFDIO islets, but nothealthy islets secreted proinsulin, even in low glucose conditions, andproinsulin secretion ceased after IL-22 treatment (FIG. 7B).

These experiments demonstrate that IL-22-IL-22R1 signaling is requiredfor normal islet function, and that exogenous IL-22 suppresses chronicER stress in HFDIO islets, reducing GSIS to normal levels while blockingproinsulin release.

Materials and Methods Acute Er-Stress Experiments

Healthy islets were cultured with 10 μg/mL tunicamycin or 5 μMthapsigargin t IL-22 for 6 hours. Alternatively the islets were culturedin the presence of ER stress-inducing cytokines t IL-22 for 24 hours.The cultures were then cultured consecutively for 30 minutes in 2.8 mMglucose, 20 mM glucose, or a combination of 20 mM glucose and 100 nMGLP-1.

High Fat Diet Induced Obesity (HFDIO) Experiments

All mice were housed in sterilized, filter-topped cages in aconventional clean facility. Six to eight week old C57BL/6 male micewere fed ad libitum on a high fat diet containing 46% of availableenergy as saturated fat, 34% carbohydrate, 20% protein (Specialityfeeds, SF04-027). Or on normal chow containing less than 10% saturatedfat (GOLDMIX, 126575). Cages of mice were randomly allocated to theexperimental groups by random draw. Investigators were not blinded totreatment by there were no subjective assessments made.

Insulin and Proinsulin Measurements

Total insulin and proinsulin were measured in supernatant from isletcultures or in serum from mice using commercially obtained ELISA kitsfrom MILLIPORE and MERCODIA, respectively. The molar insulinconcentration was determined by subtracting the molar proinsulinconcentration from molar total insulin, and then the serumproinsulin:insulin ratio was calculated.

Example 4 ER Stress Modulating Cytokines in Diabetic Pancreatic B-Cells

Although the cytokines found to be the most potent β-cell ER stressors,IL-23, IL-24 and IL-33, have not previously been implicated in T2Dpathogenesis, their mRNA were significantly increased in pancreaticβ-cells from mice with HFDIO (FIG. 8A). Similarly, this was also thecase in β-cells harvested from mice with diabetes due to leptin receptordeficiency (FIG. 8C). The IL-22-regulated pro-oxidant genes andanti-oxidant genes were increased in β-cells from both murine modelsindicating oxidative stress and a consequent anti-oxidant response(FIGS. 8B,D).

Analysis of a large transcriptome study comparing human healthy isletswith those from a patient with type 2 diabetes revealed that IL-24 andIL-33 are the most highly upregulated ER stress-modulating cytokinegenes in human type 2 diabetes. This demonstrates the clinical relevanceof the identified cytokines (Table 5).

TABLE 5 Gene Fold- Rank symbol Gene title change P-value 6 IL1RL^(#)1Interleukin 1 receptor-like 1 1.91 0.0004 (IL-33 receptor) 22 IL33Interleukin 33 1.61 0.0037 26 IL24 Interleukin 24 1.60 0.0032 57IL1R2^(#) Interleukin 1 receptor, type II 1.50 0.00005 (IL-1β decoyreceptor) 70 IL1R1^(#) Interleukin 1 receptor, type 1 1.47 0.00002 88IL22RA1 Interleukin 22 receptor, alpha 1 1.40 0.0019 Data extracted fromNCBI GEO Accession No. GSE41762; P-values are from t-tests, and arenon-adjusted; ^(#)IL1RL1, IL1R2 and IL1R1 are in a gene cluster onchromosome 2q12.

Materials and Methods Leptin Deficiency Model of Diabetes

Twenty-week old male diabetic db/db C57BL/KsJ (Lepr^(+/+)) andnon-diabetic db/h C57BL/KsJ (Lepr^(+/−)) mice were sacrificed and bloodwas collected via cardiac puncture. Db/db mice develop severe metabolicdefects in the context of type 2 diabetes (hypertension, hyperlipidemia,obesity, insulin abnormalities). A small number of animals that died forreasons unrelated to the treatment in these long-term experiments wereexcluded from the analysis. All experiments were approved by theUniversity of Queensland Animal Experimentation Ethics Committee or theAlfred Medical Research and Education Precinct Animal Ethics Committee,and conducted in accordance with guidelines set out by the NationalHealth and Medical Research Council of Australia.

Example 5 Neutralization of ER Stress-Inducing Cytokines orAdministration of Il-22 Improves Glycemic Control in Obese Mice

The in vitro findings suggested that neutralizing the ER stressorcytokines, or administering IL-22, would reduce pancreatic oxidative andER stress and improve insulin biosynthesis in obesity. In mice fed a HFDfor 15 weeks, two weeks of IL-22 administration, or antibodyneutralization of either IL-23 or IL-24, substantially reversed glucoseintolerance as measured by an i.p. glucose tolerance test (IPGTT), withIL-22 the most efficacious (FIG. 9A,D,G). The area under the curve (AUCglucose) for the IPGTT was 2.5-fold higher in mice with HFDIO and theincrease was reduced by 42, 50 and 82% with anti-IL-23, anti-IL-24 andIL-22 treatment, respectively. Improved glucose tolerance could occurvia improved insulin sensitivity. However, insulin tolerance tests(ITTs) demonstrated that treated mice had unresolved insulin resistance(FIGS. 9B, E, H). Fasting serum total insulin concentrations were2.8-fold higher in HFDIO compared to non-obese mice (FIGS. 9C, F, I andTable 6). All three therapies decreased fasted total insulinconcentrations and AUC insulin, however AUCs were greater than in micefed normal chow, consistent with requiring more insulin to deal withpersisting insulin resistance (FIGS. 9C, F, I). Anti-IL-23 and, moreeffectively, IL-22 decreased the fasted serum proinsulin:insulin ratio(FIG. 93), with IL-22 reducing serum proinsulin by 92% (Table 6).

TABLE 6 Fasting Concentrations Serum Serum insulin proinsulin Bodyweight Blood glucose concentration concentration (% original) (mmoL/L)(ng/mL) (pmol/L) HFDIO Experiment 1 10 weeks on NCD 113 ± 4  7.6 ± 1.8 0.7 ± 0.01 Not determined 16 weeks on NCD 120 ± 9  9.6 ± 0.9 0.9 ± 0.1Not detectable 16 weeks on NCD + IL-22 118 ± 3  8.9 ± 0.8 0.5 ± 0.1 Notdetectable 16 weeks on NCD + αIL-23 117 ± 3 10.1 ± 0.7 0.4 ± 0.2 Notdetectable 16 weeks on NCD + αIL-24 115 ± 3  9.5 ± 1.9 0.4 ± 0.3 Notdetectable 10 weeks on HFD 134 ± 8 10.5 ± 1.5 2.0 ± 1.2 Not determined16 weeks on HFD 147 ± 8 10.6 ± 0.5 2.5 ± 1.2 73.9 ± 32.9 16 weeks onHFD + IL-22 129 ± 9  8.8 ± 0.9 1.1 ± 0.5 9.5 ± 3.8 16 weeks on HFD +αIL-23 149 ± 7 10.7 ± 2.6 1.6 ± 0.6 22.7 ± 19.6 16 weeks on HFD + αIL-24148 ± 8  9.6 ± 1.2 1.3 ± 0.7 40.6 ± 18.2 HFDIO Experiment 2 22 weeks onNCD 140.6 ± 6  1.03 ± 0.6 0.9 ± 0.1 3.3 ± 0.6 22 weeks on HFD  225 ± 1014.1 ± 1.3 3.1 ± 1.0 152.0 ± 41.8  22 weeks on HFD + IL-22  188 ± 1311.3 ± 0.5 0.7 ± 0.4 14.1 ± 2.9  (20 ng/g) 22 weeks on HFD + IL-22  189± 17 10.6 ± 1.3 0.5 ± 0.3 4.7 ± 1.8 (100 ng/g)

Improvements in glycemic control were paralleled by decreased totalpancreatic ER stress (FIGS. 9K and 10A). Immunostaining revealed thatGrp78, which accumulates with misfolded proteins, was 11.2-fold higherin the β-cell region of HFDIO islets, and reduced by 75% after IL-22treatment (FIG. 9L and FIG. 11). β-cell insulin stores decreased by 53%in HFDIO islets, and returned to levels in non-obese mice after IL-22treatment (FIG. 9L); whereas proinsulin increased by 10.4-fold in HFDIO,and decreased by 64% after IL-22 treatment (FIG. 9L and FIG. 11). Therewas little change in the MafA transcription factor, which drives β-cellsecretory gene expression or insulin (In2) mRNA expression in HFDIO orwith treatment (FIG. 10B-D). Despite the proinsulin accumulation therewas no change in expression of the prohormone convertase that processesproinsulin into insulin (FIG. 10E). However, pancreatic inflammation wasreduced by the cytokine neutralization and IL-22 treatments (FIG. 12).

Interestingly, mice treated with IL-22, but not mice treated with IL-23or IL-24 neutralizing antibodies, progressively lost weight duringtreatment without changing food consumption (FIG. 13). Treated normalchow fed mice did not show any significant change in weight or metabolicmeasurements, although there were trends toward lower fasted bloodglucose and insulin concentrations (Table 2), and improved glucosetolerance with IL-22 (FIG. 13). IL-22 also affects hepatocytes, andhepatic ER stress occurs in obesity. Whereas hepatic Xbp1-splicing wasonly slightly increased in HFDIO, Hspa5 was highly upregulated anddecreased substantially after treatment with anti-IL-23, and anti-IL-24and IL-22 (FIG. 14).

Materials and Methods Histology, Immunofluorescence Microscopy andHistological Quantification HFDIO Experiment 1

From 13-16 weeks of the diet, mice were treated with 20 ng/g i.p.recombinant murine IL-22 twice weekly (R&D SYSTEMS), 3 μg/g anti-IL-23weekly (ELI-LILLY), 0.1 mg/mouse anti-IL-24 weekly (PROTEIN TECH), or0.5 mg/mouse irrelevant isotype control antibody, sacrificed andsampled.

HFDIO Experiment 2

From 18-22 weeks of diet, mice were treated (for 30 days) with 20 or 100ng/g i.p. recombinant murine IL-22 twice weekly (R&D SYSTEMS) or 0.1mg/mouse irrelevant isotype control antibody, sacrificed and sampled.

Fasted i.p. glucose tolerance tests (2 g/kg glucose) and insulintolerance tests (0.25 U/kg insulin; Humalog, Lilly) were performed atthe times indicated in the Figure Legends.

Pancreatic tissue was fixed in 10% neutral buffered formalin andprocessed using standard histological techniques. Standardimmunohistochemical and immunofluorescent staining methods were used todetermine the presence of Grp78, insulin, glucagon, IL-22R1, proinsulinand ATF6-GFP.

The area in pixels/mm² was determined for Grp78 and insulin(immunofluorescence) and proinsulin (immunohistochemisty) in the core ofβ-cell rich area of 3-4 islets per mouse (n=6-10 mice) using IMAGEJsoftware version 1.45s. Live cell imagine in MIN6N8 cells was carriesout at 37° C. in 5% CO₂ in a chamber using an OLYMPUS XCELLENCE RTmicroscope.

Example 6 Prolonged IL-22 Therapy Restores Insulin Sensitivity afterRestitution of Glycemic Control

To assess longer and higher dose therapy in more advanced disease micewere administered IL-22 at 20 ng/g (the first experiment dose) or 100ng/g for the last 30 days of a 22-week HFD. Body weight progressivelydecreased beginning one week into IL-22 treatment (FIG. 15A),accompanied by changes in distribution of adipose tissue with increasedepididymal and brown fat (FIG. 15G). Demonstrating rapid restoration ofglycemic control, random fed blood glucose concentration decreased tolevels seen in normal chow fed mice within 7 days with both doses ofIL-22 (FIG. 15B). An IPGTT on day 25 of therapy showed glycemic controlindistinguishable from control non-obese mice (FIG. 15C). At day 16 oftherapy, an ITT showed no or modest improvement in insulin resistance(FIG. 15D), however, by day 29 insulin resistance had decreased withboth doses of IL-22 (FIG. 15E).

Measurement of serum total insulin during day 25 IPGTT showed completesuppression of fasting hyperinsulinemia and normalization of GSIS (FIG.15F). These improvements were accompanied by normalization of the serumproinsulin:insulin ratio, with serum proinsulin decreased 97% by 100ng/g IL-22 (FIG. 16A, Table 6). The dose dependent improvement in serumproinsulin was mirrored by decreases in proinsulin staining andincreases in total insulin staining of islets (FIG. 16B).

Materials and Methods

Experiments were performed as described for previous examples with theintervention being HFDIO Experiment 2.

Example 7 Islets from IL-22 Treated Mice Show Normalized Secretion ofHigh Quality Insulin

Ex vivo culture of islets from obese mice treated with IL-22 for 30 daysdemonstrated correction of insulin hyper-secretion in response toglucose (FIG. 17A), replicating the result of ex vivo exposure to IL-22in HFD islets. When exposed to glucose/GLP-1, equal amounts of totalinsulin were secreted by HFD, HFD plus IL-22, and non-obese islets (FIG.17A). However, while no detectable proinsulin was secreted by non-obeseislets, the HFD islets secreted proinsulin, and this was reduced in adose-dependent manner by in vivo treatment with IL-22 (FIG. 17B). Thus,both ex vivo and in vivo treatment with IL-22 reduced total insulin andproinsulin secretion by islets from mice on a HFD, consistent with theamelioration of serum hyperinsulinemia and reduced serum proinsulinobserved in vivo.

To assess the functional quality of insulin compared the ability of theinsulin secreted by islets in response to glucose/GLP-1 to stimulateuptake of fluorescent deoxy-glucose (6-NBDG) by 3T3 cells differentiatedinto adipocytes. Insulin secreted by HFDIO islets ex vivo showed a 70%reduction in stimulation of adipocyte 6-NBDG uptake compared to insulinsecreted by islets from normal chow fed mice. In contrast, there wasonly a 11% reduction in adipocyte 6-NBDG uptake with insulin secreted byislets from mice treated with 100 ng/g IL-22 (FIG. 17C). Thus, in vivoIL-22 therapy improved the efficacy of secreted insulin.

Materials and Methods Glucose Uptake Experiments

3T3 cells were differentiated into adipocytes as previously described,and all experiments were carried out on day 8 post differentiation.1×10⁴ cells were seeded in a black 96-well plate and cultured in serumfree 2.8 mM glucose media prior to the addition of insulin. Cells weretreated for 1h with 2 ng/mL recombinant insulin or total insulin fromislet secretions at 37° C. as described in figure legends. Subsequently,cells were incubated with6-(N-(7-nitrobenx-2-oxa-1,3-diazol-4-yl)amino)-6-deoxyglucose (6-NBDG)(MOLECULAR PROBES) for 20 min before cells were washed with PBS andglucose uptake was determined using a POLARstar Omega plate reader.

Example 8 Effect of IL-22 Treatment on Expression of Oxidative StressRegulatory Genes and Reduces ER Stress and Inflammation

Islets from mice with HFDIO showed an upregulation of anti-oxidant genesand Nos2, and IL-22 therapy further increased expression of anti-oxidantgenes and suppressed Nos2 (FIG. 18A), emulating when β-cells and isletswere treated with IL-22 in vitro. ER stress decreased in islets fromHFDIO mice treated with both doses of IL-22, with Xbp1 splicingindistinguishable from normal chow islets with 100 ng/g IL-22 (FIG.18B). Although in the first experiment IL-22 was shown to suppress totalpancreatic inflammation, a remaining question was whether IL-22suppression of ER stress reduced inflammation within islets. Expressionof multiple cytokines and chemokines decreased in HFDIO islets from micetreated with IL-22 in vivo, with the higher dose generally moreefficacious (FIG. 18C). MIP2α mRNA reduced substantially in HFDIO isletscultured in IL-22 (FIG. 18D), paralleling decreased ER stress (FIG.18D). These observations are consistent with IL-22 therapy in obese micedisrupting the cycle of islet ER stress and inflammation.

Example 9 Effect of IL-22 Treatment on Oxidative Stress in Human IsletsCultured In Vitro

Based of these results, it is hypothesized that the inflammatorycytokines that induce ER stress in secretory cells do so as part ofrapid immune responses to viral infection. Acute ER stress induces aPERK-mediated block in protein translation, which would effectivelyreduce viral replication. Accordingly, as IL-22 prevents cytokineinduced oxidative and ER stress in secretory cells, including in primaryhuman bronchial epithelial cells, it is hypothesized that exogenouslyadministered IL-22 would increase viral replication in respiratoryepithelial cells.

To test this hypothesis mice were infected with pneumovirus (PVM), amurine respiratory virus similar to the respiratory syncytial virus(RSV) that infects the human lung. Five-day old neonatal C57BL/6 micewere administered either recombinant mouse IL-22 (rmIL-22) or anirrelevant IgG1 mock treatment. At 7 days of age, mice were infectedwith 100 PFU of PVM (i.n.) and euthanized on 1, 5 and 6 dayspost-infection. Sham-infected naïve control mice were sampled on days 1,5 and 6. Lung tissue was removed, and divided into lobes forhistological analysis and mRNA quantification. The experiment wasstopped on day 6 as 7/9 mice from the rmIL-22 treatment group diedbetween days 5 and 6 and the remaining 2 exhibited signs of suffering(FIG. 20A). PVM gene expression measured by PCR (to quantify viralnumbers) increased 2.2-fold in mice treated with IL-22 at 5 days postinfection (FIG. 20B). Consistent with increased viral replication, lunginjury was also greater at 5 days post infection in IL-22-treated mice,which also exhibited a lung injury score 1.7 times higher (FIG. 20C).Additionally, there were large areas of leukocyte infiltration in thesmall airways, most likely neutrophils in the IL-22 treated mice at day5 post-infection (FIG. 21A). Immunohistochemical detection of PVM inboth the control infected mice and the IL-22-treated infected miceshowed that PVM was located in epithelial cells, and supported thehigher viral titers measured by PCR (FIG. 21B). Thus, systemicallyadministered IL-22 has the potential to exacerbate the severity of someviral infections in humans.

Taken together with the risks associated with the functional activity ofIL-22 in tissues other than the pancreas, directly targeting IL-22 topancreatic β-cells will decrease exposure to IL-22 in the skin,intestine and liver, and therefore lower the risk of adverse effects.

Materials and Methods Administration Environment During Murine ViralInfection

Five-day old neonatal C57BL/6 mice were administered intraperitoneal(i.p.) injections of either recombinant mouse IL-22 (rmlL-22) (20 ng/gevery three days) [R&D Systems, #582-ML/CF], or an irrelevant IgG1 mocktreatment control (12.5 μg/mouse, weekly) [Lilly Pharmaceuticals,LSN2404993]. At 7 days of age, isoflurane-anesthetized mice wereinfected with 100 PFU of PVM (i.n.) and euthanized on 1, 5 and 6 DPI bysodium pentobarbital overdose (N=2-9 mice/group). Mice were euthanizedon day 6 instead of day 7 as 7/9 mice from the rmIL-22 treatment groupdied between days 5 and 6 and the remaining 2 exhibited signs ofsuffering. Sham-infected controls (naïve control) were sampled on days1, 5 and 6. Lung tissue was removed, and divided into lobes forimmunohistochemistry (left lobe fixed in 10% formalin and embedded inparaffin, superior lobe frozen in OCT (TISSUE TEK)), mRNA quantification(inferior lobe snap frozen) and protein quantification (middle andpost-caval lobes snap frozen).

Assessment of Lung Injury and Inflammation

Paraffin embedded left lobes were sectioned onto Superfrost plus slides.Sections were deparaffinized using SolV21C (MURABAN) and hydrated usinggraded ethanol solutions. Sections were stained to determine lung injuryusing hematoxylin (SIGMA ALDRICH) and eosin (SIGMA ALDRICH) (H&E)staining and were mounted on coverslips with Pertex (MEDITE). A scoringsystem for lung inflammation and injury was devised. This scoring systemassessed inflammation in the small airspaces, peribronchiolarinterstitial, alveolar interstitial and injury in the airway epithelium.The degree of lung injury and inflammation was scored blindly.

Cells infected by PVM were visualized by immunohistochemistry usingrabbit anti-mouse PVM antibody (polyclonal at 1:8000) (ALPHA DIAGNOSTIC,PVMNP14-S). Sections were deparaffinized using xylene and hydrated usingethanol. Antigen retrieval was achieved by boiling in citrate buffer (pH6) for 20 min. Sections were then blocked with 3% H₂O₂/PBS and 10%KPL/PBS 0.05% Tween. Primary antibody was incubated in 10% KPL/PBS 0.05%Tween overnight at 4° C., washed, then incubated for 45 min in donkeyanti-rabbit IgG 1:200 (JACKSON), horse-radish peroxidase conjugatedsecondary antibody before reacting with Diaminobenzidine (DAKO) for 3min. Sections were then counterstained in hematoxylin, dehydrated in agraded ethanol series and mounted onto coverslips with Pertex. PVMinfection was quantified by counting number of infected cells in 4-5bronchioles per mouse and expressed as the number of infected cells perlength of bronchiole epithelium. All sections were visualized on aVS120-L100 slide scanner microscope (OLYMPUS) and analyzed using OlyVIAv2.4 (OLYMPUS) and IMAGEJ v1.45s.

Example 10 Effect of IL-22 Treatment on Oxidative Stress in Human IsletsCultured In Vitro

Human islets express high levels of IL-22R1. To demonstrate that the ERstress-inducing or -reducing cytokines would have similar effects onhuman islets, human pancreatic islets were obtained from a single organdonor and cultured for 2 hours in the presence of ER stress-inducingcytokines or the FFA palmitate, with or without 30 min prior exposure toIL-22. IL-22 protected human islets from the production of ROS and NOinduced by IFN-γ, IL-18, IL-23, IL-24 and palmitate (FIG. 22). In humanislets IL-18 induced greater production of ROS than IL-23 and IL-24,whereas IL-24, and to a lesser extent IL-23, induced greater productionof NO than IL-18 (FIG. 22). Importantly, just 2 hours exposure to anIL-22 receptor-blocking antibody resulted in high production of both ROSand NO (FIG. 22), demonstrating that endogenous IL-22 signaling is animportant component of normal islet physiology in humans, as it was inmice. These results show that the primary effect of IL-22 on mouseβ-cells that resulted in the favorable responses to IL-22 therapy indiabetic mice, are replicated in human cells supporting theapplicability of IL-22 therapy directed at the pancreas in humandiabetes.

Materials and Methods Human Islet Culture

Human islets were recovered from organ donors and placed into overnightculture in DMEM with 2.8 mM glucose and 10% FCS. Islets were then washedand transferred into serum free DMEM for 6 h prior to experiments toassess induction of oxidative stress. Islets were then cultured for 2-24hours in the presence of 50 ng/mL of IFNγ, IL-1β, IL-23 or IL-24, or thefree fatty acid palmitate at 0.5 mM, with or without exposure to 50ng/mL IL-22 beginning 30 min prior to the introduction of the stressinducing cytokines or lipids. To test whether endogenous IL-22 affectshuman islet physiology the islets were treated for 2-24 hours with ananti-IL-22R1 (receptor) antibody at 10 μg/mL. ROS and nitrite weremeasured using the DCFDA and Greiss assays as described above for murineislets, and quantitative RT-PCR used to assess gene expression.

SUMMARY OF THE EXAMPLES

The experimental evidence presented herein demonstrates IL-22 to be apowerful endogenous paracrine suppressor of oxidative and ER stress inpancreatic islets, and that in obesity-induced hyperglycemia IL-22therapy restores glucose control by attenuating defects in pancreaticinsulin biosynthesis and secretion. IL-22 prevents oxidative and ERstress in pancreatic β-cells, regardless of whether the stress isinduced by lipids, inflammatory cytokines or environmental ROS, viaSTAT1- and STAT3-mediated upregulation of anti-oxidant genes andsuppression of oxidative stress-inducing genes. In obese mice IL-22administration had multiple highly desirable physiological consequencesincluding restoration of glucose tolerance, resolution ofhyperinsulinemia and hyperproinsulinemia, restitution of insulinsensitivity, decreased body weight and redistribution of fat to healthydepots. These findings demonstrate that in diabetes, therapy with IL-22will control hyperglycemia and preserve β-cells, and that targetingIL-22 to a β-cell will provide effective therapy while avoiding possibleadverse effects of non-directed systemic IL-22 therapy.

Although the expression of the unique element of the IL-22 receptorheterodimer, IL-22R1, is most highly expressed in the pancreas, highexpression of is also seen in the skin, intestine and liver (human geneexpression from GTex is shown is FIG. 19). However, in the experimentsdescribed above, with administration of up to 100 μg/kg of IL-22 for upto 30 days no pathology was observed in the skin, intestine or liver.However, several lines of evidence suggest that chronic high dose IL-22therapy would lead to pathology in these tissues.

High concentrations of IL-22 are found in the skin during inflammationin psoriasis and antagonism of IL-22 alleviates psoriatic pathology.High concentrations of IL-22 in the skin drive proliferation adding tothe development of psoriatic lesions, in part by upregulating serineproteases. IL-22-producing CD8 T cells have been found associated withtransplant-associated skin cancers and possibly contribute to squamouscell carcinoma growth or development in this setting.

IL-22 is generally thought to be a protective cytokine in the intestine,bolstering mucosal defense and driving proliferation in the intestineduring wound repair following infectious damage. However, chronic highconcentrations of IL-22, and low concentrations of the counteractingIL-22 binding protein, have been shown to contribute to hyperplasia andtumor development in the intestine in animal models.

The present inventors found that IL-22 suppressed ER stress in theliver, and others have reported favorable effects of IL-22 in mousemodels of acute alcohol-induced liver injury, ischemia-reperfusioninjury, fatty liver disease, toxin-induced liver damage and in viralhepatitis. However, IL-22 has been reported to induce hepatocyteproliferation and when IL-22 was over-expressed under the albuminpromoter in the liver in mice, there were increased rates ofcarcinogen-induced liver cancers. IL-22 is found associated with humanhepatocarcinomas, and carcinogen-induced liver cancers are reduced inIL-22^(−/−) mice.

Example 11 In-Vitro Assessment of Efficacy Human Vs Mouse IL-22 toSuppress Oxidative and ER Stress

The experiment using the murine MIN6N8 cells presented in FIG. 23Ademonstrates that human recombinant IL-22 reduces ER stress in murinecells, however ˜16-fold more human recombinant IL-22 was required tohave the same effect as recombinant mouse IL-22. Similarly using thehuman LS174T cells, ˜64-fold higher concentrations of mouse recombinantIL-22 were required to have the same effect as human recombinant IL-22in suppressing oxidative stress (as measured by nitrite production, FIG.23B).

Conclusions: Mouse and human IL-22 have 79% amino acid identity andthese experiments show they have reduced efficacy against the IL-22receptor from the alternate species. It is feasible to use human IL-22in in vitro experiments with murine cells and in in vivo experiments inmice, but considerably higher concentrations of cytokine will likely berequired to have the same effect as murine IL-22. Additional factorsthat need to be considered in the in vivo experiments include: (a) humanIL-22 may also have reduced affinity for the soluble IL-22 bindingprotein (IL-22RA2) and this may reduce inactivation and increase in vivoefficacy; (b) prolonged use of human IL-22 in mice may induce anantibody response resulting in more rapid clearance and impairedactivity.

Example 12 In-Vitro Assessment of Protection from ER and OxidativeStress by Human IL-22-IL-22-SCAB1 ScFv CONSTRUCT RELATIVE TO IL-22

FIG. 24 shows ER stress in MIN6N8 cells exposed to palmitate, or to therecombinant mouse and human IL-22 and each of the IL-22-GLP-1R ligandfusion proteins and an IL-22-single chain antibody construct targeting aβ-cell antigen (IL-22-ScaB1) at the highest concentrations used in theparallel experiments. As expected, palmitate induced ER stress butrecombinant mouse IL-22, human IL-22, the IL-22-GLP-1R ligand fusionproteins, MPBS-50, MPBS-51 and MPBS-52 (see, FIG. 36), and theIL-22-ScaB1 fusion protein (see, FIG. 34) did not cause ER stress.

FIG. 25A shows protection from ER stress in murine MIN6N8 β-cellsconcomitantly exposed to palmitate and human IL-22 or the 3 IL-22-GLP-1Rligand fusion proteins. MBPS-50 inhibited ER stress in a dose dependentmanner at a similar efficacy to a molar equivalent concentration ofhuman IL-22. MPBS-51 did not effectively suppress ER stress at any ofthe concentrations tested. MPBS-52 effectively suppressed ER stress, butshowed a somewhat unusual titration curve with lower efficacy than humanIL-22 at high concentrations and greater efficacy at lowerconcentrations.

FIG. 25b shows protection from oxidative stress in human LS174T coloncells concomitantly exposed to palmitate and murine IL-22, human IL-22or the 3 NN-IL-22-GLP-1R ligand fusion proteins. MBPS-50 inhibitedoxidative stress (nitrite production) but only toward the highest end ofthe concentration range used, being less effective than molar equivalentconcentrations of human IL-22. MPBS-51 did not effectively suppressoxidative stress at any of the concentrations tested, and in factappeared to increase palmitate-induced nitrite production at the lowerend of the concentration range. MPBS-52 effectively suppressed oxidativestress with a similar efficacy to equivalent molar concentrations ofhuman IL-22.

FIG. 26 shows a reduction in ER stress in MIN6N8 cells exposed topalmitate concomitantly with human IL-22 or the IL-22-ScaB1 fusionprotein.

Conclusions: The MPBS-50 and MPBS-52 fusion proteins have retained invitro efficacy for suppression of ER and oxidative stress, demonstratedin both murine and human cells. Interestingly, MPBS-50 was moreefficacious than MPBS-52 in the murine β-cells whereas MPBS-52 was moreefficacious in the human colon cells. Conversely, MPBS-51 appearsinactive in-vitro. IL-22-ScaB1 reduced ER stress induced by palmitatebut was at <50% potency compared to the native human IL-22.

Example 13 In-Vivo Assessment of the Clearance of Il-22 Fusion Protein

FIG. 27 shows the concentrations of recombinant mouse and human IL-22,and the 3 IL-22-GLP-1R ligand fusion proteins in serum over the first 2hours following administration. As previously documented mouse IL-22 isalmost completely cleared within 60 min of administration (not shown).Human IL-22 was cleared slightly more slowly and the 3 fusion proteinsgave slightly differing patterns of clearance. MPBS-50 was cleared withsimilar kinetics to recombinant human IL-22, whereas MPBS-51 and MPB-52showed a lower and somewhat more extended/later peak concentration.

Example 14 In-Vivo Assessment of Relative Activation of Stat3 by HumanIL-22-GLP-1R Ligand Fusions in Pancreatic Islets Vs. Other Target Tissue(Liver, Skin, Gut)

FIG. 28 shows the relative STAT3 fluorescence (determined per tissuearea) in pancreatic islets vs. liver, intestine and skin 30 min afterthe administration of each form of human IL-22. Results are expressed asa fold change in the tissue ratios relative to administration of humanIL-22. The ratio for MPBS-50 and -51 were not greater than for nativehuman IL-22 in the liver or intestine, but showed 45- and 113-fold meanincreases in STAT3 phosphorylation in the islet vs the skin compared tonative IL-22. Interestingly, although we observed no effect of MPBS-51in reducing ER/oxidative stress in vitro, it did induce STAT3phosphorylation in the pancreas as well as liver and gut suggesting itis activated in vivo, possibly via DPPIV protease-mediated cleavage. Incontrast to MPBS-50 and MPBS-51, MPBS-52 showed substantially increasedtargeting to the pancreatic islets, relative to liver (396-fold),intestine (338-fold) and skin (4,840-fold). These findings provide proofof principle that GLP-1R-targeting can enhance IL-22 activity in thepancreatic islets relative to the IL-22 target tissues that may showundesired adverse effects.

Conclusions: All three fusion proteins improved targeting to the isletsvs. skin and MPBS-52 administration resulted in very substantiallyincreased STAT3 phosphorylation in the islets vs. liver, skin andintestine. These data provide initial proof of principle thatIL-22-GLP-1R ligand fusion proteins can be used to enhance targeting ofIL-22 to the pancreatic islets with potentially enhanced therapeuticoutcomes.

Example 15 In-Vivo Assessment of Relative Activation of Stat3 by HumanIL-22-Scab1 Scfv Construct in Pancreatic Islets Vs. Other Target Tissue(Liver, Skin, Gut)

FIG. 29 shows the relative STAT3 fluorescence (determined per tissuearea) in pancreatic islets vs. liver, intestine and skin 30 min afterthe administration of human IL-22 at the highest doses of 15.4 nmoles/gand increasing concentrations of IL-22-ScaB1 (0.96-15.4 nmoles/g).Results are expressed as a fold change in the tissue ratios relative toadministration of human IL-22. The ratio for IL-22-ScaB1 at 0.96nmoles/g were not greater than for native human IL-22 at the highestconcentration in the liver, skin or intestine. However at the two higherdoses IL-22-ScaB1 showed substantially increased targeting to thepancreatic islets, at 15.4 nmoles/g relative to liver a 120-fold, in theintestine a 420-fold and in the skin a 110-fold increase was observed.These findings provide proof of principle that targeting (by multiplemethods) can enhance IL-22 activity in the pancreatic islets relative tothe IL-22 target tissues that may show undesired adverse effects.

Conclusions: IL-22-ScaB1 proteins improved targeting to the islets vs.other IL-22RA1 expressing tissue such as liver, skin and intestine.These data provide initial proof of principle that IL-22-fusion withantibody proteins can be used to enhance targeting of IL-22 to thepancreatic islets with potentially enhanced therapeutic outcomes.

Example 16 Dose-Finding Efficacy Study in Mice with High Fat DietInduced Obesity with IL-22-GLP-1R Ligand Fusion Proteins

This stage of testing involved a two-week therapy across a range of fivedoses (0.06-15.4 nmoles/g) of human IL-22 and the IL-22-GLP-1R ligandfusion proteins in mice that had been on a high fat diet (HFD) for 27weeks. After two weeks glycemic control was assessed by anintraperitoneal glucose tolerance test (IPGTT; all doses compared to HFDshown in FIG. 30). FIG. 31B shows that the IL-22-GLP-1R ligand fusionproteins are more potent at a lower dose in lowering IPGTT AUC comparedto native IL-22, and reduce AUC of the IPGTT effectively to thatobserved in normal chow diet (value of 1). Doses of IL-22 or theIL-22-GLP-1R ligand fusion proteins >0.96 nmoles/g gave a mean reductionin the increased area under the curve (AUC) of the IPGTT of the HFD vsnormal chow fed control mice of >50% (FIG. 31B).

Below this dose the three IL-22-GLP-1R ligand fusion proteins remainedactive whereas the efficacy of human IL-22 diminished substantially(see, FIG. 32). The increased efficacy of the IL-22-GLP-1R ligand fusionproteins is consistent with improved targeting of IL-22 to thepancreatic islets (FIG. 28). Body weight changes were also assessedduring treatment, revealing trends toward increased body weight loss atthe highest doses of the IL-22-GLP-1R ligand fusion proteins, whereasthere was little change in body weight with human IL-22 (FIG. 33). Thesefindings provide encouraging proof of principle of the increasedefficacy of the IL-22-GLP-1R ligand fusion proteins vs. native IL-22 forrestoring glycemic control in obesity/diabetes.

Material and Methods for Examples 11 to 16 Production of his-ScaB1ScFv-Il22 Human-Myc (Referred to as Il-22-Scab1)

Fusion Protein Sequence Design:

The first requirement for the development of a fusion protein able tospecifically target pancreatic β-cells was to select a targeting moietyto connect to the human IL-22 sequence. The ScaB1 single chain variablefragment (scFv) was identified as a potential means to facilitatetargeting. DNA sequences for the scFv were sourced from WO 2010/096930.The coding sequence was modified slightly to remove parts that wereunnecessary, such as extra restriction enzyme sites, linker extensions,signal peptides and artefacts (e.g. -AAA at C-terminal end of thesequence in WO 2010/096930) relating to the original method use indeveloping the scFv (see, FIG. 34A).

Human IL-22 sequences were identified using the NCBI and Uniprotdatabases:http://www.ncbi.nlm.nih.gov/gene?Cmd=DetailsSearch&Term=50616; andhttp://www.uniprot.org/uniprot/Q9GZX6. The final human IL-22 sequenceused excluded the signal peptide included in all online resources as itspresence would result in cleavage of the fusion protein during mammalianexpression (FIG. 34B). Sequences were then connected by means of asingle G4S linker to form a scFv-cytokine fusion protein with aN-terminal 6×His- and a C-terminal myc-tag to aid in protein detectionduring product development (FIG. 34C).

The initial fusion protein coding sequence was optimized by removingcodons that could cause unwanted effects such as additional cuts in thesequence when performing routine restriction enzyme digests for cloningpurposes. The designed DNA sequence was optimized for expression inChinese hamster ovary (Cricetulus griseus) cells and included a 5′leader sequence (MGWSCIILFLVATATGVHS [SEQ ID NO:293]) from the publishedMAB Xpress system and a Kozak sequence for initiation of translation.

Molecular Biology:

The fusion protein was ordered through GeneArt using their custom genesynthesis service and subsequently sub-cloned into pcDNA 3.1(+) usingHindIII and XbaI restriction sites (New England Biolabs, NEB). Colonieswere grown on LB agar plates using ampicillin resistance as selectivepressure for positive pcDNA3.1 (+) colonies. Presence of the insertedfusion protein DNA sequence was confirmed through routine colony PCR andsubsequent Sanger sequencing using T7-Forward (TAATACGACTCACTATAGGG [SEQID NO:294]) and BGH-reverse (TAGAAGGCACAGTCGAGGC [SEQ ID NO:295])primers that have sites 5′ and 3′ to the insert site on the vectorbackbone. Sequence alignment performed using the online tool, ClustalOmega, confirmed presence of the fusion protein sequence. A large scaleE. coli expression (ampicillin selection) was then completed to produceenough DNA to purify (Macherey-Nagel, NucleoBond® Xtra Midi kit) and usefor mammalian expression in suspension adapted CHO cells.

Mammalian Expression:

Aseptic techniques were maintained during cell culture protocols andcompleted in Class II biosafety cabinets, routinely cleaned using 80%(V/V) ethanol and UV sterilized. Media was prepared in the cabinets andheated to 37° C. in a water bath located external to the cabinet butstill within an area dedicated to large-scale quality proteinproduction. Transient expression of the fusion protein was performedusing PEI-mediated transfection, whereby DNA and PEI are diluted inOptiProSFM, mixed and added to suspension adapted CHO cells inchemically defined CD-CHO (Gibco, Life Technologies) media supplementedwith 8 mM Glutamax. Transfected cells were initially kept at 37° C.,7.5% CO2 and 130 rpm. Following a 5-hour incubation time a feed composedof CD-CHO+8 mM Glutamax, 0.4% (v:v) anti-clumping agent (ACA), 7.5%(v:v) each of Feed A and Feed B (Gibco, Life Technologies) was added tothe cells in the biosafety cabinet to dilute the cultures 1:2 (v:v) in1L shaker flasks (Corning), as previously described in Codamo et al.(2011).

Protein Purification:

Culture supernatants were harvested 10 days post-transfection bycentrifugation then filtered using a 0.22 μm bottle top filter(Corning). The pH was adjusted to 7.3 and supernatant stored at 4° C.until purification could be completed. As the scFv included a variableKappa (VK) subgroup I chain, this provided the means to purify thefusion protein using pre-packed HiTrap protein L columns (GE HealthcareLife Sciences) rather than the more time consuming process ofimmobilized metal affinity chromatography (IMAC) which binds His-tagsfor purification. The protocol included 15 mM NaOH for sterilization andcleaning, 1×Dulbecco's PBS (Life Technologies) as wash and runningbuffer, and 0.1 M Glycine pH 3.0 for protein elution. Eluted protein wassubsequently desalted into 1×Dulbecco's PBS using a HiPrep desaltingcolumn (GE Healthcare Life Sciences). Following purification thepurified product was run under reduced and non-reduced conditions on aNuPAGE® Novex® 4-12% Bis-Tris Protein Gel alongside the SeeBlue Plus2pre-stained protein marker (Life Technologies). The results showed thatthe purification protocol was effective in producing quality amounts ofpure fusion protein with no visible contaminants (FIG. 35). Furtheranalytical experiments are yet to be completed to determine final yieldquality.

IL-22-GLP-1R Ligand Fusions Proteins:

These fusion proteins were made by Novo Nordisk A/S (Bagsvaerd, Denmark)and their sequences are provided in FIG. 36.

In-Vitro Experiments

Cell culture: Murine MIN6N8 cells were tested negative for mycoplasma.Cells were routinely cultured in phenol-red free DMEM (LifeTechnologies) containing 25 mM glucose (3.4 g/L sodium bicarbonate, 50U/mL penicillin and streptomycin, 71.5 μM β-mercaptoethanol and 10% heatinactivated fetal bovine serum) and transferred to DMEM as above with5.5 mM glucose 48 h prior to experimentation. Human LS174T cells werecultured in DMEM containing 10% heat-inactivated fetal bovine serum, 50U mL⁻¹ penicillin and streptomycin.

ER Stress Measurement: MIN6N8 cells were transfected with the ERAIreporter plasmid (F-XBP1ΔDBD-venus, using Lipofectamine 2000 accordingto the manufacturer's instructions. For ERAI there was negligiblefluorescence in the absence of ER stressors, but upon induction of ERstress, splicing of XBP1 mRNA by IRE1a results in the translation ofVenus but not an active form of XBP1. Fluorescence was measured(excitation: 485 nm; emission: 520 nm) using a POLARstar Omega platereader and cells were treated 48 h after transfection. 48 hpost-transfection cells were exposed to 0.5 mM palmitate to induce ERstress and concomitantly treated with mouse or human IL-22 over a rangeof concentrations (0.12-15.4 nM). After 24 h, ERAI-sXBP1 GFP reporterfluorescence was measured.

Oxidative Stress Measurement: Cells were exposed to 0.5 mM palmitate toinduce ER stress and concomitantly treated with mouse or human IL-22over a range of concentrations (0.12-15.4 nM) for 2 h. The GriessReagent Kit for Nitrite Determination was performed according to themanufacturer's instructions (Molecular Probes) to assess theintracellular levels of nitrite (the stable metabolite of nitric oxide)using lysates of β-cells prepared in RIPA buffer (150 mM NaCl, 1% NP40,0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 7.5).

In-Vivo Animal Experiments:

Targeting to IL-22 to pancreas: Non-obese male C57BL/6 at 6-8 weeks ofage. Each IL-22-GLP-1R ligand fusion protein or hIL-22 was administeredi.p. at 256 ng/g of IL-22 and the molar equivalents for the individualfusion proteins (n=3 per regimen). IL-22-ScaB1 was administered at 15.4nmoles/g, 3.8 nmoles/g and 0.96 nmoles/g. Control mice received a PBScontrol i.p. injection only.

To assess clearance of IL-22 and IL-22-GLP-1R ligand fusion proteinsblood samples were collected before injection and at 15, 30, 45, 60, 90and 120 min. Serum concentrations of IL-22 and the IL-22-GLP-1R ligandfusion proteins were determined using a human IL-22 double-determinantELISA (Biolegend; 434505) against a standard curve for the individualfusion proteins.

To assess in vivo activation of the IL-22 receptor signaling pathway,after 30 min the mice were sacrificed and the following tissues weresnap frozen and sections were prepared for each mouse: pancreas, skin(ear, tail), colon (rolled), and liver. Sections were stained byimmunofluorescence for phosphorylated STAT3 using the Phospho-Stat3(Tyr705) (D3A7) antibody.

Intraperitoneal Glucose Tolerance Test:

C57BL/6 male mice from 6-8 weeks of age were fed a high fat dietcontaining 46% of available energy as saturated fat, 34% carbohydrate,20% protein (Speciality feeds, SF04-027) for 27 weeks and treated asfollows:

IL-22-GLP-1R ligand fusion protein or IL-22 alone were administeredtwice weekly i.p. at the following doses: 1, 4, 16, 64 and 256 ng/g ofIL-22 (0.12-15.4 nmoles/g) and the molar equivalents for IL-22-GLP-1fusion proteins (n=3 per regimen, except for the lowest dose where n=2);control mice received PBS control only. A fasted Intraperitoneal glucosetolerance tests (IPGTT) was performed (2 g kg−1 glucose) on day 14 whenthe experiment was terminated (tissues were not kept for analysis). Bodyweight was recorded on days 0, 5, 10 and 13.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. Those of skill in the artwill therefore appreciate that, in light of the instant disclosure,various modifications and changes can be made in the particularembodiments exemplified without departing from the scope of the presentinvention. All such modifications and changes are intended to beincluded within the scope of the appended claims.

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What is claimed is:
 1. A method of treating a metabolic disorder of theliver, comprising administering a therapeutic agent comprising an IL-22polypeptide and an antigen-binding molecule, wherein the IL-22polypeptide is fused or otherwise conjugated, directly or indirectly, tothe antigen-binding molecule, and the antigen-binding molecule is anantibody or antigen-binding fragment that comprises heavy chain CDRsequences comprising a CDR1 of SEQ ID NO: 62, a CDR2 selected from thegroup consisting of SEQ ID NOs: 63 to 67, and a CDR3 selected from thegroup consisting of SEQ ID NOs: 68 to 71; and light chain CDR sequencescomprising a CDR1 of SEQ ID NO: 72, a CDR2 selected from the groupconsisting of SEQ ID NOs: 73 to 77, and a CDR3 selected from the groupconsisting of SEQ ID NOs: 78 to
 82. 2. The method of claim 1, whereinthe antibody or antigen-binding fragment comprises heavy chain CDRsequences comprising a CDR1 of SEQ ID NO: 62, a CDR2 of SEQ ID NO: 63,and a CDR3 of SEQ ID NO: 68; and light chain CDR sequences comprising aCDR1 of SEQ ID NO: 72, a CDR2 of SEQ ID NO: 73, and a CDR3 of SEQ ID NO:78.
 3. The method of claim 1, wherein the antibody or antigen-bindingfragment comprises a heavy chain variable sequence comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 47 to51; and a light chain variable sequence comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 52 to
 56. 4.The method of claim 1, wherein the antibody or antigen-binding fragmentcomprises a heavy chain variable sequence comprising the amino acidsequence of SEQ ID NO: 47 and a light chain variable sequence comprisingthe amino acid sequence of SEQ ID NO:
 52. 5. The method of claim 1,wherein the antigen-binding molecule is a single chain antibody.
 6. Themethod of claim 1, wherein the IL-22 polypeptide comprises the aminoacid sequence of SEQ ID NO:
 4. 7. The method of claim 1, wherein theantigen-binding molecule is fused or otherwise conjugated, directly orindirectly, to the C-terminus of the IL-22 polypeptide.
 8. The method ofclaim 1, wherein the antigen-binding molecule is fused or otherwiseconjugated, directly or indirectly, to the N-terminus of the IL-22polypeptide.
 9. The method of claim 1, wherein the antigen-bindingmolecule is conjugated to the IL-22 polypeptide via an interveninglinker.
 10. The method of claim 9, wherein the linker is a peptide. 11.The method of claim 10, wherein the peptide linker comprises the aminoacid sequence GGGGS.
 12. The method of claim 1, wherein theantigen-binding molecule is conjugated to the C-terminus of the IL-22polypeptide via a peptide linker.
 13. The method of claim 1, wherein theantigen-binding molecule is conjugated to the N-terminus of the IL-22polypeptide via a peptide linker.
 14. The method according to claim 1,wherein the antigen-binding molecule is a single chain antibodycomprising heavy chain CDR sequences comprising a CDR1 of SEQ ID NO: 62,a CDR2 of SEQ ID NO: 63, and a CDR3 of SEQ ID NO: 68; and light chainCDR sequences comprising a CDR1 of SEQ ID NO: 72, a CDR2 of SEQ ID NO:73, and a CDR3 of SEQ ID NO: 78, and wherein the antigen-bindingmolecule is conjugated to the C-terminus of the IL-22 polypeptide via apeptide linker.
 15. The method according to claim 1, wherein thetherapeutic agent comprises the amino acid sequence of SEQ ID NO: 285.16. The method of claim 1, wherein the metabolic disorder of the liveris non-alcoholic steatohepatitis (NASH).
 17. The method of claim 1,wherein the metabolic disorder of the liver is non-alcoholic fatty liverdisease (NAFLD).